in vitro transcription 
 reactions , particularly those mediated by 
 t7 rna polymerase , have become a cornerstone of modern rna biochemistry 
 and biophysics .
these cell - free transformations facilitate the preparation 
 of short as well as long native rna transcripts using synthetic and 
 plasmid - derived dna templates .
while of rather general utility , t7 
 rna polymerase requires a specific promoter for optimal transcription 
 and tends to also be rather sensitive to sequence composition , particularly 
 next to its consensus promoter .
numerous studies have analyzed the 
 initiation and elongation stages in these processes and , practically , 
 identified optimal sequences at the transcript s
guanosine residues are frequently found at positions + 1 and + 2 , 
 where altered sequences typically suffer from significantly diminished 
 transcription efficiency . in vitro prepared transcripts 
 therefore almost exclusively possess a gtp at their 5 end , 
 with 15 out of 17 reported promoters initiating transcription with 
 pppg and 13 with pppgpg .
a formidable 
 contemporary challenge , which is compounded by the 
 constraints outlined above , is the need to modify rna transcripts , 
 typically with fluorescent probes , for diverse biochemical and biophysical 
 applications .
this problem has been tackled in several ways , including the development 
 of orthogonal base pairing systems , which require , however , the synthesis 
 of modified dna templates in addition to the necessary complementary 
 modified triphosphates . the fastidious behavior of t7 rna polymerase has 
 also limited the modification position , with most appearing remote 
 to the promoter , where the enzymatic process is believed to be beyond 
 its vulnerable initiation phase . replacing the initiating gtp at position + 1 with alternatives such 
 as gmp and guanosine ,
some with modifications on the monophosphate 
 or ribose , has been explored with different levels of success .
transcription initiation with gtp analogs , where the heterocyclic 
 nucleus has been altered has not , to our knowledge , been explored . 

this has motivated the study reported here , where newly synthesized gtp , a highly isomorphic and emissive analog , has been investigated 
 as a gtp surrogate in t7 rna transcription reactions .
the recent 
 completion of an emissive rna alphabet , a fluorescent 
 ribonucleoside set comprised of highly emissive purine and pyrimidine 
 analogs , all derived from thieno[3,4-d]pyrimidine , presents unique opportunities for the generation 
 of modified rna constructs . despite the high structural resemblance 
 to their native counterparts ,
it was 
 unclear at the onset of this project whether or not polymerases could 
 accommodate and effectively incorporate these modified nucleosides 
 into oligonucleotides .
as discussed above , 
 particularly challenging is the initiation of in vitro transcription 
 reactions with gtp analogs , which is inevitable in this context . in 
 this contribution
we critically assess the ability of t7 rna polymerase 
 to initiate rna transcription and elongate the nascent modified transcript 
 using gtp ( 2 ) , a highly emissive and isomorphic 
 gtp analog , and compare the results to its performance with the native 
 triphosphate .
we demonstrate that the modified nucleotide is capable 
 of initiating and maintaining transcription reactions , leading to 
 the formation of fully modified and highly emissive transcripts .
importantly , 
 to assess proper folding and function of rna transcripts where all g residues have been replaced with a synthetic analog , 
 we explore modified hammerhead ribozymes , catalytic rna assemblies , 
 which are likely to be exceedingly sensitive to such alterations . 
 the fully modified enzyme and substrate of the hammerhead ribozyme 
 are efficiently transcribed , and the impact of replacing all g residues 
 with g is evaluated .
we further demonstrate that the 
 emissive transcripts can be used to monitor the ribozyme - mediated 
 cleavage reaction in real time .
the 5-triphosphate of g was synthesized from 
 the parent nucleoside using freshly distilled 
 pocl3 and tributylammonium pyrophosphate ( scheme 1 ) .
the triphosphate 
 was purified by ion - exchange chromatography and reverse - phase hplc 
 ( see experimental section ) .
final treatment 
 with chelex 100 afforded the analytically pure nucleotide [ p nmr  9.98 ( d , j = 20.1 hz , p ) , 10.64 ( d , j = 18.6 hz , p ) , 22.64 ( t , j = 18.6 hz , p ) ] . reagents and conditions : ( i ) 
 pocl3 , ( meo)3po , 0 c ; ( ii ) tributylammonium 
 hydrogen pyrophosphate in dmf , bu3n , 0 c .
transcription reactions with the analytically pure gtp ( 2 ) and t7 rna polymerase were performed 
 to first 
 analyze its enzymatic incorporation into short rna oligonucleotides . 

a short dna promoter  template duplex was used to discern the ability of gtp to initiate 
 transcription and be incorporated during the elongation phase ( figure 1 ) .
the dna template 3 is terminated 
 with a single t at the 5 end so a lone a is directed to the 
 3 end of the transcript .
when trace amounts of -p atp are used , only successfully transcribed full length 
 labeled rna products would be visible , whereas short failed transcripts 
 would be undetected after gel electrophoresis ( figure 1 ) .
t7 promoter and template 3 depicting the enzymatic 
 incorporation reaction using natural ntps and gtp or gtp resulting in transcripts 4 or 5 .
the t7 promoter and template 3 were annealed , and 
 transcribed in the presence of natural ntps or with gtp 
 replacing gtp and a trace of -p atp .
a phosphorimage 
 revealed a full length 10-mer product ( transcript 5 ) 
 using gtp that corresponded to the natural triphosphates 
 transcript 4 ( figure 2a ) .
the 
 overall yield of transcript 5 , containing four g residues , compared to the natural unmodified transcript 4 was 70  3% , indicating that the average individual incorporation 
 was 91  1% .
next , a large - scale transcription reaction was run 
 and uv shadowing was used to visualize all products . comparing the 
 transcription reactions with gtp to reactions with gtp 
 illustrates that the desired product and abortive transcripts appear 
 almost identical ( compare lanes 1 and 2 , respectively , in figure 2b ) .
importantly , when visualizing the gel under 
 uv illumination ( 302 nm ) , the product and initiation phase truncated 
 transcripts are highly fluorescent ( right figure 2b ) . following extraction ,
the isolated yield of the g modified 10-mer ( transcript 5 ) was 78  12% compared 
 to the native 10-mer ( transcript 4 ) , averaging 94  
 2% per g incorporation .
the transcripts were characterized 
 by esi ( figure s1a ) and digested using 
 s1 nuclease and dephosphorylated .
the nucleoside mixtures were subjected 
 to hplc - ms analysis . comparing the chromatogram obtained for the native 
 10-mer ( 4 , figure s3b ) to 
 that of the modified one ( 5 , figure 3 )
( a ) small scale transcription using trace -p atp . lane 1 : control transcription reaction in the absence 
 of gtp or gtp . lane 2 : control reaction in the presence 
 of all natural ntps .
lane 3 : reaction in the presence of equimolar 
 concentration of gtp and gtp .
incorporation efficiencies of gtp transcripts are reported with respect to transcription in the 
 presence of gtp .
all reactions were performed in triplicate , and the 
 errors reported are the standard deviations .
( b ) large scale transcription 
 reaction using template 3 with all natural ntps ( lane 
 1 and 1 ) or atp , utp , ctp , and gtp ( lanes 2 and 
 2 , 78  12% isolated yield ) with uv light at 254 nm ( on 
 tlc plate ) and 302 nm ( pl ) .
hplc - ms traces of the ( a ) mixture of nucleosides used as a standard 
 and ( b ) digestion results of transcript 5 .
digestion 
 of 12 nmol of transcript was carried out using s1 nuclease 
 for 2 h at 37 c and followed by dephosphorylation with alkaline 
 phosphatase for 2 h at 37 c .
the ribonucleoside mixture obtained 
 was analyzed by reverse - phase analytical hplc , using a mobile phase 
 of 06% acetonitrile ( 0.1% formic acid ) in water ( 0.1% formic 
 acid ) over 12 min ; flow rate 1 ml / min . to test the runoff transcription of longer constructs and 
 assess 

the function of the resulting transcripts , longer dna templates ( 6 and 7 ) were used to generate hammerhead ribozymes : 
 the natural all native substrate ( s ) , the modified substrate ( g - s ) , the natural enzyme ( e ) , and the modified enzyme ( g - e ) ( figure 4 ) .
these transcription 
 reactions were executed only on a large scale , and the rna transcripts 
 ( s , g - s , e , and g - e ) were isolated after 
 polyacrylamide gel electrophoresis ( figure s4 ) , characterized by esi ( figure s1b and s1c ) , and then digested ( figure s3c and s3d ) .
as before , the full length products and all short failed transcripts 
 in transcription reactions using gtp are highly emissive 
 ( figure s4 ) .
t7 promoter and templates 6 and 7 depicting 
 the enzymatic incorporation reaction using natural ntps and gtp or gtp resulting in transcripts s , thg - s , e , or thg - e .
g is underlined and bolded blue in the transcripts g - s and g - e .
following purification , 
 the natural substrate ( s ) and g - substrate ( g - s ) were dephosphorylated with alkaline phosphatase and 5 
 labeled with t4 polynucleotide kinase according to standard protocols .
the assembled ribozymes were then tested for 
 the anticipated strand cleavage in all different combinations ( figure 5 ) , using conditions similar to those previously 
 published , where the ribozyme cleavage reaction 
 was initiated by mixing equal volumes of buffered solutions containing 
 mgcl2 of substrate with enzyme .
the reactions contained 
 excess enzyme to obtain pseudo first - order kinetic rate constants 
 ( k2 ) .
single turnover reactions 
 at 31 c contained 0.3 m substrate ( including a trace 
 of 5-p labeled material ) and 3 m enzyme , 
 in 50 mm tris - hcl ph 7.0 , 200 mm nacl , and 10 mm mgcl2 . 

the rate constants obtained for the native hh ribozyme s and e ( figure 5a ) and the substrate - modified one g - s 
 and e ( figure 5b ) were 0.15  0.1 min and 0.12  0.1 min , respectively 
 ( figure 6b ) .
the unmodified hh enzyme e cleaved 
 87% and 86% of the substrates s and g - s , respectively , 
 at 20 min . the fully modified g - containing enzyme ( g - e ) showed no cleavage of s or g - s at 31 c 
 ( figures 5c , 5d , and 6a ) .
reactions of g - e with the natural 
 and modified substrates at slightly elevated temperatures ( 37 c ) 
 did show a small amount of sequence specific cleavage , estimated at 
 about 2% after 40 min ( figure s5 ) .
hammerhead 
 ribozymes and cleavage reactions of ( a ) natural substrate 
 and enzyme ( s and e ) , ( b ) modified substrate and natural enzyme ( g - s and e ) , ( c ) natural substrate and modified enzyme ( s 
 and g - e ) , and ( d ) modified substrate and modified enzyme 
 ( g - s and g - e ) .
( a ) hh ribozyme cleavage reaction results were followed by p radioactive labeling of substrate strands s and g - s . 

s and p1 , and g - s and g - p1 indicate substrate and product strands ( figure 5 ) .
all reactions were conducted at 31 c and contained 
 0.3 m substrate ( including a trace of 5-p labeled material ) , 3 m enzyme , 50 mm tris ph 7.0 , 200 mm 
 nacl , and 10 mm mgcl2 .
the reactions were quenched at the 
 given times ( t in min ) and resolved by gel electrophoresis 
 on a denaturing 20% polyacrylamide gel with 7 m urea .
( b ) initial 
 kinetics of s and e and g - s and e. the pseudo - first - order 
 rate constants ( k2 ) of the cleavage reactions 
 are determined as the slope of ln(fraction cleaved ) versus time .
( c ) 
 ribozyme - mediated cleavage curves as determined by p 
 data for s and e and g - s and e. fraction cleaved ( s / s0 ) was determined by dividing the amount of cleaved substrate 
 by the sum of the full length and cleaved substrate .
the cleavage of g - s by the native 
 enzyme e was monitored 
 with nonradiolabeled material using steady - state fluorescence spectroscopy 
 under the same conditions as the experiments performed with the radiolabeled 
 constructs but in a slightly larger volume ( see figure s6 for absorption and emission spectra of g - s ) . mixing the substrate with the enzyme in a fluorescence cuvette 
 at 31 c gave final concentrations of 0.3 m substrate 
 and 3 m enzyme in 50 mm tris - hcl ph 7.0 , 200 mm nacl , and 10 
 mm mgcl2 .
the fluorescence intensity , monitored at 450 
 nm , increased during the reaction ( figure 7a and 7c ) .
alternatively , the fully modified 
 enzyme g - e mixed with the native substrate s , which showed 
 no measurable strand cleavage at 31 c when monitored using the p labeled substrate , displayed minimal fluorescence intensity 
 changes ( figure 7b and 7d ) .
the fraction cleaved of g - s from the radioactive 
 and fluorescence experiments was normalized and showed a similar trend 
 ( figure s7 ) .
importantly , page analysis 
 confirms the presence of two fluorescent products g - p1 and g - p2 ( see figure s8 ) .
fluorescence spectra of ( a ) g - s and e ( blue ) 
 ( excitation 
 470 nm , emission 425485 nm , slit widths 8 nm ) and ( b ) s and g - e ( black ) ( excitation 470 nm , emission 425485 nm , 
 slit widths 4 nm ) , where t = 0 min and t = 20 min spectra have thicker lines .
all reactions were conducted 
 at 31 c and contained 0.3 m substrate , 3 m enzyme , 
 50 mm tris ph 7.0 , 200 mm nacl , and 10 mm mgcl2 . the fluorescence 
 intensity shown at 450 nm over 20 min of the ( c ) cleavage of g - s by e ( blue ) and ( d ) mixing of s and g - e 
 ( black ) .
as 
 has been previously demonstrated , fluorescent nucleoside analogs 
 can tremendously facilitate the study of nucleic acid folding , recognition , 
 and catalysis , including the enablement of real time assays .
of particular significance is the modification of oligonucleotides 
 with isomorphic nucleoside analogs , as their high similarity to their 
 canonical counterparts and nonperturbing nature frequently results 
 in faithful folding and function .
a case 
 in point is our recent study of emissive mrnas , where specific g residues 
 have been surgically replaced by g using solid - phase 
 synthesis .
these modified rna constructs 
 were recognized by the ribosome and capable of fluorescently reporting 
 discrete steps in translation . here
we 
 set to explore the ability of gtp to support the initiation 
 step of in vitro transcription reactions and then elongate the nascent 
 rna transcripts to yield full length products in which all g residues 
 are replaced by g. we then evaluate the impact of this 
 rather dramatic modification on the function of the hh ribozyme as 
 a prototypical functional rna system .
the commonly used t7 promoters 
 end with c , so one could not test 
 a single g or g incorporation in g - containing transcripts 
 where a g residue is remote to the promoter .
we therefore initially 
 used a short oligonucleotide 3 that has been previously 
 used by our lab to evaluate t7 rna polymerase s ability to 
 produce short modified transcripts ( figure 1 ) .
transcription reactions using template 3 in the presence of gtp , ctp , atp , and utp but 
 no gtp ( figure 2a , lane 3 ) yielded full length 
 products , which suggested that gtp could indeed initiate 
 such transcription reactions .
the high incorporation efficiency of 
 the g modification at an average yield of 91  1% 
 per incorporation demonstrated its structural and functional similarity 
 to g. it is likely , however , that the incorporation yield at positions 
 + 1 and + 3 is lower than those at positions + 6 and + 8 , due to their 
 proximity to the promoter .
capitalizing on these positive results , 
 the transcription reactions were scaled up to provide large quantities 
 of full length products for analytical characterization .
indeed , ms 
 analysis and enzymatic digestion reactions followed by hplc - ms analysis 
 further confirmed that gtp was indeed recognized by t7 
 rna polymerase , and efficiently utilized during the initiation and 
 elongation phases ( figure 3 ) .
the intense fluorescence 
 of the abortive transcripts provided another positive indication that gtp was indeed the agent responsible for initiating transcription 
 ( figure 2b ) .
dna templates ( e.g. , 6 and 7 ) , yielding 
 medium rna transcripts , also demonstrated that gtp is 
 efficiently incorporated during the initiation and elongation phases . 

much longer transcripts , which include 
 multiple g residues at the 5-end , as well as 
 several consecutive g residues , have also been transcribed 
 in high yields , with an average incorporation yield of 95% per gtp ( see figure s9 ) .
we submit 
 that these observations validate g as a true isomorphic 
 nucleoside analog of g , which is faithfully recognized by the polymerase , 
 while retaining high selectivity for wc pairing .
although , in principle , 
 multiple alterations within an rna transcript might be functionally 
 detrimental , the intense emission of these per - modified transcripts 
 suggests potential utility ( see below ) . additionally , established 
 protocols in rna biochemistry allow ligations of rna fragments into 
 larger constructs , which suggest that short g - containing 
 rnas can be ligated to longer native ones for certain applications .
in addition to gtp being a substrate for t7 rna polymerase , 
 we tested the fully modified oligonucleotides , specifically g - s and g - e , for their ability to function as components 
 in a hh ribozyme , as a prototypical catalytically active rna .
the 
 substrates ( s and g - s ) were first radioactively labeled 
 to visualize their ribozyme - mediated cleavage . incidentally , this 
 successful labeling demonstrates that the 5-end of the transcript g - s does not hamper alkaline phosphatase - mediated dephosphorylation 
 and t4 polynucleotide kinase - mediated phosphorylation .
these observations 
 are of significance , as they suggest that modifying rna transcript 
 with g does not hinder enzymatic transformations by commonly 
 used molecular biological agents .
the unmodified hh enzyme e 
 cleaves its native substrate s and the 
 fully modified one g - s with similar rates , indicating 
 that replacing all g residues with g in the hh substrate 
 does not substantially interfere with ribozyme catalysis .
in contrast , the 
 fully modified hh enzyme g - e showed very little cleavage 
 ability of either the native substrate s or the corresponding modified g - s .
this suggests that the substitution of g for g interferes with either the folding or catalysis of the hh enzyme . 

as the modified substrate is almost fully duplexed and effectively 
 cleaved by the native enzyme , attention is then focused on the relevant 
 residues in the enzyme that are proposed to be involved in catalysis : 
 g8 , g10.1 , and g12 ( figure 5c ) . in the proposed 
 ribozyme cleavage mechanism ,
the n7 of g10.1 coordinates a divalent 
 metal ion , which appears to be involved 
 in the phosphodiester cleavage reaction .
the lack of the imidazole 
 ring and hence of n7 in g may therefore explain the severely 
 attenuated catalytic activity of the fully modified enzyme g - e .
additionally , the nucleobase of g12 is involved in the cleavage 
 reaction where the putatively deprotonated n1 position acts as a base 
 to abstract the proton on the o2 of c17 , which then nucleophilically 
 attacks the adjacent 3 phosphate , eventually leading to strand 
 cleavage . since the pka of the n1 position of g within the folded ribozyme 
 likely differs from that of g
the sugar and not the nucleobase is proposed to be 
 involved in catalysis , making it less likely that modification 
 at this position is directly hindering cleavage .
importantly , the cleavage of the modified hh substrate g - s by the native enzyme e can be observed using steady - state 
 fluorescence 
 spectroscopy , demonstrating the utility of such emissive transcripts 
 for monitoring rna related processes in real time ( figure 7a and 7c ) . a significant 
 fraction of the large increase seen in fluorescence intensity likely 
 originates from the cleavage of g - s by e because inactive 
 combinations ( such as that of s and g - e ) exhibit much 
 smaller fluorescence intensity changes ( figure 7b and 7d ) .
we note , however , that changes 
 in emission intensity observed when mixing such fluorescent rna strands 
 represent multiple events , including the annealing , folding , and mg coordination , as well as strand cleavage and dissociation . 

this is supported by the small fluorescence increase seen when the 
 s and g - e are mixed to generate an inactive hh ribozyme , 
 which likely reflect only conformational changes and metal coordination 
 ( figure 7b and 7d ) . 

notably , however , when the normalized data generated by p labeling is compared to the fluorescence - generated rates for g - s and e , similar overall trends and rates are seen ( figure s6 ) .
while these  deficiencies  
 can likely be circumvented by monitoring the process using a stopped - flow 
 kinetic apparatus ( thus separating the fast events from the relatively 
 slow cleavage reaction ) , we emphasize that monitoring catalytic rnas 
 using simple benchtop steady - state fluorescence spectrometers can 
 be an effective way to probe substrate cleavage and inhibition .
it greatly reduces the overall experimental 
 time , and unlike p - monitored reactions , which are not 
 monitored in real - time , can provide additional insight into conformational 
 changes , hybridization , and magnesium binding events , among others .
gtp is found to be accepted by t7 polymerase as a 
 faithful gtp surrogate .
this highly isomorphic nucleoside triphosphate 
 can initiate transcription , as well as be incorporated during the 
 elongation phase in short and longer oligonucleotides .
importantly , 
 such modified transcripts , which contain a pppg at their 
 5-end , are also successfully dephosphorylated with alkaline 
 phosphatase and then phosphorylated with t4 polynucleotide kinase , 
 illustrating that other terminus - modifying enzymes tolerate this modification 
 as well . to investigate the impact of g substitution 
 on rna function , we tested hh ribozyme combinations where the substrate 
 and enzyme were per - modified with g. a hh ribozyme containing 
 a fully modified substrate g - s hybridized to a native 
 enzyme e undergoes efficient phosphodiester bond cleavage that can 
 be monitored either with radioactively labeled substrate followed 
 by page or , in real time , using gs fluorescence . 

in contrast , the fully modified hh enzyme g - e displayed 
 very little cleavage ability with either s or g - s .
for example , the inactivity of the fully g modified hh enzyme suggests utility of this nucleoside 
 in probing specific mechanistic questions in rna catalysis .
moreover , due to their reliable hybridization 
 and wc pairing , highly isomorphic but potentially inactive hh constructs 
 could provide useful tools for structural analysis .
as such , the observation that gtp is accepted 
 by t7 polymerase as a faithful gtp surrogate ( in both its initiation 
 and elongation phases ) and that other enzymes , commonly used in molecular 
 biology , accept such per - modified strands as viable substrates opens 
 up numerous creative opportunities to utilize such modified nucleosides 
 and oligonucleotides , both as structural as well as fluorescent tools . 

even 2-aminopurine , an extensively employed emissive a 
 isoster , fails to perform in certain cases .
we 
 feel , however , that g can be an extremely useful probe 
 due to its unique structural features and favorable photophysical 
 characteristics .
oligonucleotides were purified 
 by polyacrylamide gel electrophoresis and desalted on sep - pak ( waters 
 corporation ) .
ntps 
 and the ribonuclease inhibitor ( ribolock ) were obtained from fermentas 
 life science .
radiolabeled -p atp ( 10mci / ml , 
 3000 ci / mmol ) and -p atp ( 10mci / ml , 6000 ci / mmol ) 
 were obtained from perkinelmer .
chemicals for preparing buffer solutions 
 were purchased from fisher biotech ( enzyme grade ) . autoclaved
0.1% 
 depc treated water was used in all biochemical reactions and fluorescence 
 titrations .
small molecule mass spectra ( ms ) were recorded 
 at the university of california , san diego , chemistry and biochemistry 
 mass spectrometry facility , utilizing an agilent 6230 hr - esi - tof mass 
 spectrometer .
reverse - phase hplc ( vydac c18 column ) purification and 
 analysis were carried out using an agilent 1200 series instrument . 

steady - state fluorescence 
 experiments were carried out in a microfluorescence cell ( 125 l ) 
 with a path length of 1.0 cm ( hellma gmbh & co. kg , mllheim , 
 germany ) on a horiba jobin yvon ( fluoromax-3 ) spectrometer .
mass spectra 
 for oligonuceotides were obtained on a thermofinnigan lcq deca xp 
 at trilink biotechnologies , inc .
tris(tetrabutylammonium ) 
 hydrogen pyrophosphate ( 0.99 g , 1.1 mmol ) in a 10 ml round - bottom 
 flask , and g ( 60 mg , 0.20 mmol ) in a 25 
 ml round - bottom flask , were separately coevaporated with anhydrous 
 pyridine and dried .
trimethyl phosphate ( 2 ml ) was added to g and cooled in an ice bath to 0 c .
phosphoryl chloride ( 46 
 l , 0.5 mmol ) was added slowly , and the reaction was stirred 
 for 2 h at 0 c , resulting in a pinkish brown solution .
the coevaporated 
 tris(tetrabutylammonium ) hydrogen pyrophosphate was dissolved in 2 
 ml of anhydrous dmf and added to the g reaction mixture . 

then tributyl amine ( 0.26 mmol , 1.1 mmol ) was added and the reaction 
 was kept stirring at 0 c for 40 min . to the reaction mixture 
 was added 6 ml of 1 m triethylammonium bicarbonate buffer ( teab ) , 
 and the mixture was stirred briefly .
the mixture was then transferred 
 to a separatory funnel and washed with 10 ml of etoac .
the organic 
 layer was then back - extracted with 5 ml of 1 m teab .
the aqueous layers 
 were combined and concentrated under reduced pressure at room temperature 
 to afford an oily yellow residue .
the residue was dissolved in 10 
 ml of 0.05 m ammonium bicarbonate buffer and loaded onto a deae sephadex 
 a25 anion - exchange column kept in a cold room at 4 c .
the column 
 was eluted using a gradient mixer with 0.050.5 m of ammonium 
 bicarbonate buffer .
a fraction collector was used to collect 260 fractions 
 that were about 8 ml ( 220 drops ) .
the fractions containing the triphosphate 
 were evaporated under reduced pressure at 10 c , and then the 
 residue was lyophilized . to purify the triphosphate further , the residue 
 was run on another deae sephadex a25 anion - exchange column and eluted 
 with 0.060.6 m ammonium bicarbonate buffer .
after lyophilization , 
 the triphosphate was treated with 25 mg of chelex 100 for 15 min with 
 occasional shaking , and then filtered .
the triphosphate was further 
 purified by hplc ( phenomenex synergi fusion - rp 80a c18 column , 4 m , 
 250  10 nm , 04% acetonitrile in 50 mm teab buffer , ph 
 6.0 , 30 min ) .
h nmr ( 500 mhz , d2o )  8.10 ( d , j = 4.7 hz , 1h ) , 5.164.97 
 ( m , 1h ) , 4.34 ( s , 1h ) , 4.21 ( dd , j = 11.5 , 9.5 hz , 
 4h ) ; p nmr ( 202 mhz , d2o )  9.98 
 ( d , j = 20.1 hz , p ) , 10.64 ( d , j = 18.6 hz , p ) , 22.64 ( t , j = 18.6 hz , p ) ; hr esi - ms ( negative ion mode ) [ c11h15n3o14p3s ] calculated 537.9493 , found 537.9500 ; esi - ms ( negative 
 ion mode ) [ c11h15n3o14p3s ] calculated 538.24 , found 537.98 
 and 559.96 as [ m  2h + na ] .
single strand dna 
 templates were annealed to an 18-mer t7 rna polymerase 
 consensus promoter sequence in te buffer ( 10 mm tris - hcl , 1 mm edta , 
 100 mm nacl , ph 7.8 ) by heating a 1:1 mixture ( 10 m ) at 90 
 c for 3 min and cooling the solution slowly to room temperature . 

transcription reactions were performed in 40 mm tris - hcl buffer ( ph 
 7.9 ) containing 500 nm annealed templates , 10 mm mgcl2 , 
 10 mm dithiothreitol ( dtt ) , 10 mm nacl , 2 mm spermidine , 1 u/l 
 rnase inhibitor ( ribolock ) , 1 mm gtp or 1 mm gtp , 1 mm 
 ctp , 1 mm utp , 20 m atp , 2 ci -p 
 atp ( 800 ci / mmol stock ) , and 2.5 u/l t7 rna polymerase ( fermentas ) 
 in a total volume of 20 l .
after 3 h at 37 c , reactions 
 were quenched by adding 10 l of loading buffer ( 7 m urea in 
 1 tbe with 0.05% bromophenol blue and 0.05% xylene cyanol ) , 
 heated to 75 c for 3 min , and 10 l was loaded onto an 
 analytical 20% denaturing polyacrylamide gel .
transcription efficiencies 
 are reported with respect to transcription in the presence of natural 
 nucleotides .
transcription efficiencies were determined from three 
 independent reactions , and the errors reported represent standard 
 deviations . to preparatively isolate rna and for enzymatic 
 digestions 
 large scale transcription reactions using template 3 were 
 performed in a 250 l reaction volume under similar conditions , 
 with the following changes .
the reaction contained 1 mm atp , ctp , 
 and utp , 1 mm gtp or gtp , 15 mm mgcl2 , 500 
 nm template , 1500 units t7 rna polymerase , and 250 units of ribolock . 

after incubation for 5 or 6 h at 37 c , the precipitated magnesium 
 pyrophosphate was removed by centrifugation .
the mixture was heated at 
 75 c for 3 min and loaded onto a preparative 20% denaturing 
 polyacrylamide gel .
the gel was uv shadowed ; appropriate bands were 
 excised , extracted with 0.5 m ammonium acetate , and desalted on a 
 sep - pak column .
concentrations of the rna transcript were determined 
 using absorption spectroscopy at 260 nm using the following extension 
 coefficients : c , 7200 ; u , 9900 ; g ; 11500 ; a , 15400 ; and g , 5517 lmol cm . to preparatively isolate rna and for 
 enzymatic digestions large scale transcription reactions using template 6 and 7 were performed in a 250 l reaction 
 volume under similar conditions , with the following changes .
the reaction 
 contained 2 mm ctp , utp , and atp , 2 mm gtp or gtp , 20 
 mm mgcl2 , 500 nm template , 6 u/l ( 1500 u ) t7 rna 
 polymerase , and 1 u/l ( 250 u ) of ribolock .
after incubation 
 for 5 or 6 h at 37 c , the precipitated magnesium pyrophosphate 
 was removed by centrifugation .
the 
 mixture was heated at 75 c for 3 min , and loaded onto a preparative 
 20% or 15% denaturing polyacrylamide gel .
the gel was uv shadowed ; 
 appropriate bands were excised , extracted with 0.5 m ammonium acetate , 
 and desalted on a sep - pak column .
concentrations of the rna transcript 
 were determined using absorption spectroscopy at 260 nm as described 
 above .
all 
 transcripts ( 12 nmol of 4 , 5 , s , g - s , e , g - e ) 
 were incubated with s1 nuclease in reaction buffer ( promega ) for 2 
 h at 37 c .
the reaction was further treated with alkaline phosphatase 
 and dephosphorylation buffer ( promega ) for 2 h at 37 c .
the 
 ribonucleoside mixture obtained was analyzed by reverse - phase analytical 
 hplc with an agilent column eclipse xdb - c18 ( 5 m , 4.6  
 150 mm ) .
mobile phase : 06% acetonitrile ( 0.1% formic acid ) 
 in water ( 0.1% formic acid ) over 12 min ; flow rate 1 ml / min .
mass spectra as raw data were taken 
 by esi mass spectrometer in negative ion mode with xcalibur software 
 version 1.3 , and the raw esi - ms m / z data were deconvoluted by promass for xcalibur version 2.5 sr-1 . 

the running buffer was 10 mm tert - butylamine in 70% 
 acetonitrile in water .
all deconvoluted mass spectra are in figure s1 and an example of raw m / z data and deconvoluted spectra are shown in figure s2 . the rna transcripts s and g - s ( 12.9 pmol and 12.3 pmol , 
 respectively ) in 3 l of 10 
 dephosphorylation buffer and 1 l of calf intestinal alkaline 
 phosphatase in a total volume of 30 l were incubated at 37 
 c for 2 h. water ( 70 l ) was added and the reaction mixture 
 extracted with 100 l of phenol : chloroform ( chcl3):isoamyl alcohol ( iaa ) = 25:24:1 . the water layer was extracted 
 with chloroform ( 100 l ) .
the rna in the aqueous layer was precipitated 
 with 6 l of glycoblue , 20 l of 10 m nh4oac , 
 and 400 l of etoh and put in dry ice bath for 1 h , followed 
 by centrifugation at 14,000 rpm for 20 min and removal of the supernatant . 

the pellet was washed 4 with 50 l of cold 70% etoh . the 
 pellet was air - dried for 30 min and then dissolved in 38 l 
 of water .
five l of 10 kinase buffer , 1 l of dithiothreitrol , 
 5 l of -p atp , and 1 l of t4 polynucleotide 
 kinase , were added and the reaction was heated to 37 c for 2 
 h. the rna was then precipitated ( 2 l of glycoblue , 10 l 
 of 10 m nh4oac , and 200 l of etoh ) and washed ( 1 
 with 25 l of cold 70% etoh ) , similar to the above procedure . 

the pellet was dissolved on 1 tbe 7 m urea loading buffer , and 
 then the rna was resolved by gel electrophoresis on a denaturing 20% 
 polyacrylamide gel .
the rna was cut out and extracted with water overnight , 
 filtered , and then concentrated using a speed vac .
cleavage reactions were 
 conducted in a total reaction volume of 34 l for the natural 
 enzyme ( e ) and 22 l for the g - enzyme ( g - e ) with the substrate ( s ) or g - substrate ( g - s ) for radiography . for the fluorescence - based experiments ,
the reactions were carried out at 
 31 c in a buffer containing 50 mm tris - hcl ( ph 7.0 ) and nacl 
 ( 200 mm ) .
buffered solutions of the substrate ( 0.6 m with traces 
 of 5-p labeled substrate ) and enzyme ( 6 m ) 
 were denatured separately by heating to 90 c for 90 s and cooled 
 to room temperature over 10 min to allow for refolding .
mgcl2 ( to make a final concentration of 10 mm ) was added to both the enzyme 
 and substrate , and both were equilibrated at 31 c for 10 min . 

the cleavage reaction was then initiated by manually mixing equal 
 volumes of the modified or natural substrate ( 0.6 m ) with the 
 enzyme or modified enzyme ( 6 m ) in a heat block at 31 c , 
 to give final concentrations of 0.3 m of the substrate and 
 3 m of the enzyme and 10 mm mgcl2 . for initial data 
 points ( time = 0 ) ,
following initiation of the reaction , 
 4 l aliquots were removed at designated time periods and quenched 
 with 12 l of urea containing loading buffer ( 7 m urea , 1 
 tbe , and 0.05% bromophenol blue , and xylene cyanol ff ) .
the tubes 
 were heated to 90 c for 90 s and loaded on a 20% polyacrylamide 
 with 7 m urea gel .
corresponding bands were quantified on a personal 
 molecular imager and analyzed with quantity one software ( biorad ) .
rate constants 
 ( k2 ) were calculated as the slope of ln(1 
  s / s0 ) versus 
 time , where s / s0 is the 
 fraction of cleaved substrate . for experiments utilizing a radioactively 
 labeled substrate ,
s / s0 was determined by dividing the amount of cleaved substrate by the 
 sum of the full length and cleaved substrates .
 the fastidious behavior of t7 rna 
 polymerase limits the incorporation 
 of synthetic nucleosides into rna transcripts , particularly at or 
 near the promoter . 
 the practically exclusive use of gtp for transcription 
 initiation further compounds this challenge , and reactions with gtp 
 analogs , where the heterocyclic nucleus has been altered , have not , 
 to our knowledge , been demonstrated . 
 the enzymatic incorporation of thgtp , a newly synthesized isomorphic fluorescent nucleotide 
 with a thieno[3,4-d]pyrimidine core , is explored . 
 
 the modified nucleotide can initiate and maintain transcription reactions , 
 leading to the formation of fully modified and highly emissive rna 
 transcripts with thg replacing all guanosine residues . 
 
 short and long modified transcripts are synthesized in comparable 
 yields to their natural counterparts . to assess proper folding and 
 function , 
 transcripts were used to assemble a hammerhead ribozyme 
 with all permutations of natural and modified enzyme and substrate 
 strands . 
 the thg modified substrate was effectively cleaved 
 by the natural rna enzyme , demonstrating the isomorphic features of 
 the nucleoside and its ability to replace g residues while retaining 
 proper folding . 
 in contrast , the thg modified enzyme showed 
 little cleavage ability , suggesting the modifications likely disrupted 
 the catalytic center , illustrating the significance of the hoogsteen 
 face in mediating appropriate contacts . 
 importantly , the ribozyme 
 cleavage reaction of the emissive fluorescent transcripts could be 
 followed in real time by fluorescence spectroscopy . beyond their utility 
 as fluorescent probes in biophysical and discovery assays , the results 
 reported point to the potential utility of such isomorphic nucleosides 
 in probing specific mechanistic questions in rna catalysis and rna 
 structural analysis . 
Introduction
Results
Discussion
Conclusions
Experimental
Section