probes capable of recognizing 
 specific mixed - sequence double - stranded 
 dna ( dsdna ) regions have been long - sought - after as they can be developed 
 into tools that enable modulation of gene expression at the transcriptional 
 level , gene editing , and detection of specific genetic signatures . 

early examples of dsdna - targeting probes include triplex - forming oligonucleotides ( tfos ) and peptide nucleic acids ( pnas ) , as well as minor groove binding polyamides .
unfortunately , only a subset of the possible target regions is available 
 for recognition by these probes due to requirements for exclusive 
 purine content ( tfos / pnas ) or short target regions ( polyamides ) .
consequentially , 
 significant efforts have been devoted to develop alternative approaches , 
 which has resulted in tfos and pnas with reduced target site limitations .
more recently , engineered proteins such as zinc 
 finger nucleases , transcription activator - like effector nucleases 
 ( talens ) and  in particular 
crispr / cas9 systems , have gained a tremendous amount of attention , 
 despite mounting concerns regarding recognition specificity and cellular 
 delivery .
another class of compounds 
 that has emerged from these efforts are the so - called pseudocomplementary 
 dna and pna ( pcdna / pcpna ) , in which a short dna or pna duplex 
 is modified to contain pseudocomplementary base pairs between 2-thiothymine 
 and 2,6-diaminopurine ( figure 1a ) .
the steric clash between the 2-thio and the 2-amino group 
 perturbs and weakens the hydrogen bonding between these moieties , 
 resulting in a destabilized probe duplex .
in contrast , 2-thiothymine 
 and 2,6-diaminopurine form stable base pairs with canonical adenine 
 and thymine , respectively . the difference in thermodynamic stability 
 between probe duplexes and duplexes between individual probe strands 
 and complementary dna ( cdna ) allows for double - duplex invasion of 
 dsdna target regions under certain conditions ( figure 1b ) .
thus , pcdna can recognize terminal target 
 regions , while pcpna also recognize internal target regions of dna 
 duplexes , albeit at low ionic strengths .
however , a recent study has 
 suggested that recognition of mixed - sequence dsdna regions by pcpna 
 may be possible under the highly viscous conditions found in the nucleus .
we , and later 
 others , have pursued an alternative strategy 
 for the construction 
 of energetically activated double - stranded probes for recognition 
 of mixed - sequence dsdna regions , which is based on forced intercalation 
 of aromatic moieties .
our invader probes are short 
 dna duplexes containing + 1 interstrand zipper arrangements of intercalator - functionalized 
 nucleotides ( figure 1c ; for a definition of the zipper nomenclature , see experimental section ) .
this motif , which we denote as an energetic hotspot , forces two intercalators into the same 
 region of the duplex , resulting in unwinding and destabilization as the nearest neighbor exclusion principle is violated .
according to this principle , the space between the 
 two nearest base pairs on either side of a bound intercalator , will 
 not be bound by a second intercalator due to limitations in local 
 helix expandability ( every intercalation event unwinds the duplex 
 by 3.4  ) , and/or to avoid 
 disruption of highly stable stacking interactions between the first 
 bound intercalator and neighboring nucleobases .
on the other hand , each of the two strands comprising an 
 invader probe display very high affinity toward cdna since the intercalators 
 stack strongly with the neighboring base pairs , acting as molecular 
 glue ( figure 1c ) .
we have previously demonstrated that the differences in thermostability 
 between invader probes and probe - cdna duplexes can drive mixed - sequence 
 recognition of isosequential dna duplexes , dna hairpins , and chromosomal dna targets .
initially , 2-n-(pyren-1-yl)-2-amino--l - lna ( locked nucleic acid ) monomers were used to construct 
 the energetic hotspots of invader probes .
however , the challenging synthesis of these 
 building blocks , prompted us to conduct a search for more feasible 
 structural and functional mimics , which , among others , resulted in 
 the identification of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x as 
 a next - generation invader modification ( figure 1a ) .
access to 
 building blocks that are based on simpler sugar scaffolds has facilitated 
 extensive structure  property relationship studies aiming at 
 improving the recognition efficiency of invader probes . in the present study
, we set out to study if the dsdna - recognition 
 potential of invader probes can be increased further through incorporation 
 of pseudocomplementary base pairs .
we hypothesized that these chimeric pseudocomplementary invader probes will be more energetically 
 labile , yet form even more stable duplexes with cdna than either canonical 
 invader or pcdna probes , leading to more favorable energetics for 
 dsdna - recognition ( figure 1b ) . toward this end
, two different approaches were followed . 
 in the first , we wanted to integrate the intercalator as part of a 
 pseudocomplementary nucleotide , which led to the identification of 
 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine monomer y as a target ( figure 1a ) .
incorporation of this monomer in a + 1 interstrand zipper arrangement 
 opposite of conventional 2,6-diaminopurine dna d monomers 
 would furnish a double - stranded probe with a pseudocomplementary energetic 
 hotspot ( dy probes ) ( figure 1b ) . in the second approach , we separated 
 the two key structural features by using conventional 2-thiothymine 
 dna monomer s and 2,6-diaminopurine dna monomer d , alongside energetic hotspots comprised of the conventional 
 invader monomer x ( dsx probes ) ( figure 1b ) .
we demonstrate 
 that the latter type of probes is particularly interesting for dsdna - recognition 
 applications .
( b ) representation 
 of energy 
 levels of different dsdna - targeting probes and the corresponding duplexes 
 with cdna ( only one probe - target duplex is shown ) .
note , 
 the large difference in energy between pc - invader : cdna duplexes and 
 pc - invader probe duplexes .
the key 2-n-(pyren-1-yl)methyl-2-n - methyl-2-amino-2-thiouridine phosphoramidite 6 was obtained from known nucleoside 1 , following 
 a similar general strategy as was used for the synthesis of 2-o-[2-(methoxy)ethyl]-2-thiothymidine ( scheme 1 ) .
nucleoside 1obtained in 61% yield over six steps from uridine  was first subjected to a sequence of 
 protecting group manipulations , i.e. , 3-o - acetylation , 5-o - detritylation and 5-o - methanesulfonylation , to afford nucleoside 2 in 55% yield over three steps .
prolonged refluxing in anhydrous 
 ethanol in the presence of sodium bicarbonate , results in the formation of 2-o - ethyluridine 
 derivative 3 in 52% yield , presumably via a nucleophilic 
 opening of an o2,o5-anhydrouridine intermediate .
subsequent 
 o5-dimethoxytritylation of 3 using standard conditions 
 affords nucleoside 4 in 88% yield , which upon treatment 
 with h2s - saturated pyridine in the presence of 1,1,3,3-tetramethylguanidine provides 2-thiouridine derivative 5 in 82% yield .
treatment of nucleoside 5 with 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite and n , n - diisopropylethylamine affords target 
 phosphoramidite 6 in 81% yield , corresponding to an overall 
 yield of 17% from nucleoside 1 .
dmtr = 4,4-dimethoxytrityl ; 
 py = pyren-1-yl ; ms = methanesulfonyl ; tmg = 1,1,3,3-tetramethylguanidine ; 
 pcl reagent =
phosphoramidite 6 was used to incorporate monomer y into oligodeoxyribonucleotides 
 ( ons ) via automated solid - phase dna synthesis .
extended hand - coupling 
 ( 15 min ) and the use of 4,5-dicyanoimidazole as an activator resulted 
 in stepwise coupling yields of 95% .
ons modified with monomer x were synthesized as previously described ( 15 min coupling , 
 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole as 
 an activator , 99% coupling yield ) .
the corresponding phosphoramidites of monomers s and d were obtained from commercial sources and incorporated into 
 ons using the conditions for incorporation of monomer y ( stepwise coupling yields > 95% ) . to prevent desulfurization in 
 ons 
 modified with y or s monomers , nucleotide 
 phosphite to phosphate oxidation
was performed using tert - butylhydroperoxide / ch3cn / h2o ( 10 min ) rather 
 than the standard aqueous iodine solution .
the identity and purity of the modified ons were established through 
 maldi - tof ( table s1 ) and ion - pair reverse - phase 
 hplc ( > 90% purity unless otherwise noted ) , respectively .
pyrene - functionalized 
 2-thiouracil monomer y was incorporated 
 into the same 9-mer mixed - sequence ons that was previously used for 
 evaluation of invader monomer x. thermal denaturation temperatures ( tm s ) of duplexes between y - modified ons and cdna 
 or crna were determined in a medium salt phosphate buffer ( [ na ] = 110 mm ) and compared relative to unmodified and corresponding x - modified duplexes .
the resulting denaturation curves display 
 the expected monophasic sigmoidal transitions ( figure s1 ) .
duplexes between y1y4 and cdna are significantly more stable than unmodified 
 reference duplexes ( tm between 
 + 2.5 and + 11.5 c , table 1 ) , whereas heteroduplexes with crna are much less stable ( tm between 6.5 to + 2.5 c , table s2 ) . ons in which y monomers 
 are flanked by 3-purines result in greater duplex stabilization 
 than ons with 3-flanking pyrimidines ( e.g. , compare tm s for y1 and y3 , table 1 ) .
this sequence 
 dependence , along with the prominent dna selectivity ( table s3 ) , is typical for ons modified with intercalating 
 pyrene moieties .
surprisingly , y - modified 
 ons form slightly less stable duplexes with cdna than their x - modified counterparts ( compare tm s of x1x4 and y1y4 , table 1 ) , which suggests that the binding modes responsible 
 for the stabilizing contributions of the pyrene and 2-thiouracil moieties 
 are not fully compatible . to generate invader probes with pseudocomplementary 
 energetic hotspots , we synthesized ons in which 2-amino-2-deoxyadenosine 
 monomers flank monomer y ( i.e. , the dy series ) . 

replacing regular 2-deoxyadenosines with d monomers 
 increases the cdna / crna affinity of y - modified ons by 
 15 c , presumably due to stabilization from the extra 
 hydrogen bond in d : t base pairs relative to normal a : t 
 pairs ( e.g. , compare tm for y1 and dy1 , table 1 and table s2 ) .
similar 
 relative increases in cdna affinity are observed when 2-deoxyadenosines 
 are replaced with d monomers in otherwise unmodified 
 ons ( see tm for d1d4 vs cdna , table 1 ) , which is in agreement with previous studies .
tm = change in tm relative to reference 
 duplex dna1:dna2 ( tm  29.5 c ) , where dna1 : 5-gtg 
 ata tgc and dna2 : 3-cac tat acg .
tm s are determined as the maximum of the first 
 derivative of melting curves ( a260 vs t ) recorded in medium salt phosphate buffer ( [ na ] = 110 mm , [ cl ] = 100 mm , ph 7.0 ( nah2po4/na2hpo4 ) ) , using 1.0 m 
 of each strand .
reported tm s are 
 averages of at least two measurements within 1.0 c ; a = adenin-9-yl 
 dna monomer , c = cytosin-1-yl dna monomer , g = guanin-9-yl dna monomer , 
 and t = thymin-1-yl dna monomer . for structures of monomers
next , we set out to study dna duplexes with different 
 interstrand 
 zipper arrangements of y monomers and dy segments to identify probe architectures that are strongly activated 
 for dsdna - recognition . as expected from our previous studies on invader 
 probes , y1:y3 , which features a + 1 interstrand zipper of y monomers , 
 is much more thermolabile than y1:y2 featuring 
 a 1 interstrand zipper ( table 2 ) , or duplexes between y - modified ons 
 and cdna ( table 1 ) . 

the destabilization is likely a consequence of the nearest neighbor 
 exclusion principle being violated . in line with 
 our hypothesis , introduction of a 2,6-diaminopurine d monomer opposite of the pyrene - functionalized 2-thiouracil y monomer ,
decreases the tm s 
 of the duplexes ( e.g. , compare tm of dy2:dy3 and y1:y2 , table 2 ) .
interestingly , 
 the destabilizing effect of the pseudocomplementary base pairs is 
 more pronounced when the y monomers are positioned in 
 a 1 zipper orientation ( drop in tm of 9.5 c from y1:y2 to dy2:dy3 , relative to drop of 2.0 c from y1:y3 to dy1:dy4 , table 2 ) .
this indicates that the pseudocomplementary 
 energetic hotspot architecture of dy1:dy4 does not fully harness the activating effects from both structural 
 elements .
the tm - based conclusions 
 were largely 
 corroborated by the gibbs free energies associated with duplex formation , 
 which were derived from denaturation curves via line fitting ( table 2 ) .
thus : ( i ) duplexes between y - modified ons 
 and cdna are much more stable than unmodified reference duplexes ( g between 16 and 7 kj / mol , 
 first and second g columns , table 2 ) due to more favorable 
 enthalpy ( h between 76 and 
 44 kj / mol , table s7 ) .
( ii ) in comparison , 
 the corresponding x - modified duplexes are slightly more 
 stable , while the d - modified duplexes are far less stable 
 ( e.g. , compare g for y1:cdna , x1:cdna and d1:cdna , table 2 ) .
( iii ) duplexes 
 between ons with dy motifs and cdna are generally less 
 stable than the corresponding y - modified duplexes ( e.g. , 
 compare g for y1:cdna and dy2:cdna , table 2 ) .
this contrasts the tm - based conclusions ( table 1 ) , but likely reflects the different entropies of these 
 duplexes ( table s8 ) , leading to different 
 temperature dependencies of the gibbs free energies .
( iv ) formation 
 of y1:y3 ( + 1 zipper ) is less thermodynamically 
 favorable than y1:y2 ( 1 zipper ) 
 ( third g column , table 2 ) .
( v ) duplexes with pseudocomplementary 
 energetic hotspots are slightly less stable than the corresponding 
 duplexes featuring only regular energetic hotspots ( e.g. , compare 
 g for dy1:dy4 and y1:y3 , table 2 ) .
the energetic nature of dy1:dy4 is the result of particularly unfavorable 
 enthalpy ( h = + 95 kj / mol , table s7 ) , likely reflecting a violation of the 
 nearest neighbor exclusion principle and concomitant formation of 
 destabilizing pseudocomplementary base pairs . ( vi )
pc - dna without 
 any energetic hotspots range between being slightly more stable to 
 slightly less stable than unmodified duplexes ( e.g. , see g for d1:y2 and y1:d3 , table 2 ) .
consequentially , + 1 zipper duplex y1:y3 is much more energetically activated than 1 
 zipper duplex y1:y2 , as gauged by the free 
 energy available 
 for recognition of isosequential dsdna targets grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb )  g ( ona : onb )  g ( dsdna ) , where ona : onb denotes a double - stranded probe and  dsdna  is the 
 isosequential dsdna target for ona : onb ( compare 
 grec293 for y1:y2 and y1:y3 , table 2 ) .
dy1:dy4 , featuring 
 a pseudocomplementary energetic hotspot , is slightly more energetically 
 activated for dsdna - recognition than y1:y3 ( compare grec293 for dy1:dy4 and y1:y3 , table 2 ) .
as expected , dy2:dy3 , which also features two pseudocomplementary base pairs but has 
 an 1 interstrand zipper arrangement of y monomers , 
 is far less activated for dsdna - recognition ( compare grec293 for dy2:dy3 and dy1:dy4 , table 2 ) .
pc - dna without energetic hotspots are also far less 
 activated for dsdna - recognition ( e.g. , compare grec293 of y1:d3 and dy1:dy4 , table 2 ) .
unexpectedly , dy1:dy4 has 
 lower thermodynamic 
 dsdna - recognition potential than conventional invader probe x1:x3 ( compare grec293 for dy1:dy4 and x1:x3 , table 2 ) , which likely is due to two factors : ( i ) intercalation of the pyrene 
 moieties perturbs the local duplex geometry , which weakens the normally 
 stabilizing base pairs between 2,6-diaminopurine : thymine ( d : t ) and 2-thiouracil : adenine ( y : a ) , leading to lower - than - expected 
 cdna - affinity of dy - modified ons ( e.g. , compare g for x1:cdna , y1:cdna and dy1:cdna , table 2 ) , and ( ii ) + 1 interstrand zipper arrangements 
 of nucleotide monomers with intercalating pyrene moieties perturb 
 local duplex geometries , which , in turn , 
 are likely to reduce the steric clash between 2,6-diaminopurine and 
 2-thiouracil normally occurring in pseudocomplementary base pairs , 
 resulting in less pronounced probe destabilization .
it is also possible 
 that the pseudocomplementary base pairs of dy1:dy4 interfere with the forced and destabilizing intercalation 
 of the two pyrene moieties ; this is supported by the uv  vis 
 absorption characteristics of dy1:dy4 . normally , 
 dna duplexes with + 1 interstrand zipper motifs of intercalating pyrene - functionalized 
 monomers exhibit markedly blue - shifted pyrene absorption relative 
 to dna duplexes with other interstrand zipper motifs , which is indicative of reduced 
 pyrene - nucleobase interactions due to 
 a locally perturbed duplex geometry ( e.g. , compare max for x1:x3 relative to x1:x2 , table 2 ) .
the same trend is observed for y1:y3 ( compare max for y1:y3 relative to y1:y2 ) , but the trend is less 
 pronounced for dy1:dy4 ( compare max for dy1:dy4 relative to dy2:dy3 ) , indicating that dy1:dy4 is not strongly perturbed
. zp = zipper . for conditions of thermal 
 denaturation , see table 1 .
grec293 ( ona : onb ) = g ( ona : cdna ) + g ( cdna : onb )  
 g ( ona : onb )  
 g ( dsdna ) .
absorption spectra were recorded at t = 10 c using each strand at 1.0 m concentration 
 in tm buffer .
data for x1:x2 and x1:x3 are from ref ( 32 ) and are included to facilitate 
 comparison .
on the 
 basis of the observed grec293 values , 
 we decided to determine the dsdna - recognition properties of y1:y3 and dy1:dy4 relative 
 to benchmark invader x1:x3 using an electrophoretic 
 mobility shift assay from our earlier studies .
thus , a digoxigenin ( dig ) labeled dna hairpin ( dh)comprised 
 of a 9-mer double - stranded mixed - sequence stem , which is linked by 
 a t10 loop  was used as a model dsdna target ( figure 2a and 2b ) .
room temperature incubation of dh1 with y1:y3 , dy1:dy4 , or x1:x3 in a hepes buffer of considerable ionic 
 strength , results in dose - dependent formation of a ternary recognition 
 complex as evidenced by the emergence of a slower migrating band on 
 nondenaturing page gels ( figure 2c ) .
response 
 curves reveal that x1:x3 , y1:y3 , and dy1:dy4 have c50 values of 0.8 m , 2.8 
 m , and 1.5 m , respectively ( figure 2d ) , which mirrors the trend 
 in grec293 values ( table 2 ) .
the binding specificities of y1:y3 , dy1:dy4 , and x1:x3 were examined by incubating a 200-fold 
 molar excess of the probes with dna hairpins dh2dh7 , which deviate in the nucleotide sequence at one position 
 relative to the invader probes ( figure 2b ) .
no recognition was observed , demonstrating that 
 invader - mediated dsdna - recognition proceeds with excellent binding 
 specificity ( figure 2e ) .
( a ) illustration 
 of recognition process ; ( b ) sequences and thermal denaturation temperatures 
 of dna hairpins with isosequential ( dh1 ) or nonisosequential 
 stems ( dh2dh7 ) ; underlined nucleotides 
 indicate sequence deviations relative to probes ; ( c ) representative 
 electrophoretograms of recognition of dh1 using 1- to 
 500-fold molar excess of y1:y3 or dy1:dy4 ; ( d ) dose  response curves ( average 
 of at least three independent experiments ; error bars represent standard 
 deviation ) ; ( e ) electrophoretograms illustrating incubation of dh1dh7 with 200-fold molar excess of x1:x3 , y1:y3 , or dy1:dy4 .
experimental conditions for electrophoretic 
 mobility shift assay : separately preannealed targets ( 34.4 nm ) and 
 probes ( variable concentrations ) were incubated for 1216 h 
 at ambient temperature in 1x hepes buffer ( 50 mm hepes , 100 mm nacl , 
 5 mm mgcl2 , 10% sucrose , 1.4 mm spermine tetrahydrochloride , 
 ph 7.2 ) and then resolved on 16% nondenaturing page ( 70 v , 2.5 h , 
 4 c ) using 0.5 tbe as a running buffer ( 45 mm 
 tris , 45 mm boric acid , 1 mm edta ) ; dig : digoxigenin .
the above results suggest that incorporation 
 of pseudocomplementary 
 energetic hotspots  i.e . , 5-yd-3:3-dy-5 cassettes 
provide less of a benefit for 
 dsdna - recognition than originally anticipated . at this stage ,
we hypothesized 
 that the two structural elements that activate invader and pc - dna 
 probes for dsdna - recognition 
the intercalator - functionalized 
 nucleotides forming the energetic hotspots , and the pseudocomplementary 
 base pairs between 2,6-diaminopurine and 2-thiouracil  need 
 to be spatially separated in order to harness their full benefits . 

accordingly , a series of 13-mer dsx - modified invader 
 probes were designed , i.e. , double - stranded probes featuring energetic 
 hotspots comprised of conventional invader building block x , along with pseudocomplementary base pairs between regular 2,6-diaminopurine d and 2-thiothymine s monomers . the following 
 probes were synthesized : ( i ) two different dsx - modified 
 invader probes , in which the energetic hotspot either is next to or 
 one nucleotide away from two pseudocomplementary base pairs ( dsx1:dsx2 and dsx3:dsx4 ) , ( ii ) an invader probe comprised of regular x monomers 
 ( x5:x6 ) , ( iii ) an invader probe with a single 
 pseudocomplementary energetic hotspot ( dy5:dy6 ) , and ( iv ) three pcdna probes , each containing two differentially 
 spaced , regular pseudocomplementary base pairs ( sd1:sd2 , sd3:sd4 and sd5:sd6 ) ( table 3 ) .
first , tm s for 
 duplexes between individual probe strands and cdna ( table 3 , first two tm columns ) or crna ( table s9 ) were determined . in line with observations from the 9-mer series , x - modified ons form highly thermostable duplexes with cdna 
 ( tm = 1112 c ) and 
 less thermostable duplexes with crna ( tm = 3 c ) . in comparison , ons with dy motifs 
 display slightly higher crna affinity ( tm for dy5/dy6 = 4.55.0 c , table s9 ) and slightly lower cdna affinity ( tm = 10 c , table 3 ) , again suggesting that the normally stabilizing 
 base - pairs between 2,6-diaminoadenosine : thymine and 2-thiouracil : adenine 
 are weakened by proximal intercalation of the pyrene moieties .
encouragingly , dsx - modified ons display significantly increased cdna and 
 crna affinity relative to x - modified ons ( tm , dna = 1415 c , table 3 ; tm , rna = 7.08.5 c , table s9 ) , suggesting that separation of the intercalators and the modified 
 nucleobases is beneficial for binding affinity . in comparison ,
regular 
 pcdna strands form far less stable duplexes with cdna ( tm , dna = 2.54.0 c , table 3 ) .
benchmark invader probe x5:x6 and dy5:dy6 are 
 both relatively thermolabile ( tm = 0.5 c and 1.5 c , 
 respectively , table 3 ) .
dsx probes are slightly less stable ( tm = 2.0 c and 3.0 c 
 for dsx1:dsx2 and dsx3:dsx4 , respectively , table 3 ) .
however , comparison with the corresponding pcdna 
 probes suggests that the full destabilizing effect of the pseudocomplementary 
 base pairs still is not fully realized with these probe architectures 
 ( e.g. , compare tm of 8.0 , 
 0.5 , and 3.0 for sd5:sd6 , x5:x6 and dsx3:dsx4 , respectively , table 3 ) .
the above tm - based conclusions 
 are 
 substantiated by the gibbs free energies for formation of duplexes 
 ( table 3 ) . as a result 
 of these stability trends ,
dsx probes are significantly 
 more thermodynamically activated for dsdna - recognition than x5:x6 , dy5:dy6 , or 
 any of the regular pcdna ( trend in grec293 values : dsx3:dsx4 < dsx1:dsx2  x5:x6 < dy5:dy6  sd5:sd6 < sd3:sd4 < sd1:sd2 , table 3 ) .
tm = change in tm relative to reference 
 duplexes dna3:dna4 ( tm  37.5 c ) , where dna3 : 5-ggta 
 tatata ggc , dna4 : 3-ccat atatat ccg .
x , y , d and s , see figure 1 . on the basis of the observed grec293 values
, we decided to evaluate the dsdna - targeting 
 efficiency of dsx1:dsx2 , dsx3:dsx4 , x5:x6 , and dy5:dy6 using a similar electrophoretic mobility shift 
 assay as used in the 9-mer series ( figure 3a ) .
thus , a dig - labeled dna hairpin ( dh8)comprised of a 13-mer double - stranded mixed - sequence 
 stem that is linked by a t10 loop  was used as a 
 model dsdna target .
incubation of dh8 with the various 
 13-mer invader probes results in dose - dependent formation of a slower 
 moving band on nondenaturing page gels ( figure 3b ) .
as expected from the initial 9-mer studies , 
 the parent invader x5:x6 recognizes dh8 more effectively at low probe concentrations than dy5:dy6 , which has a pseudocomplementary energetic 
 hotspot ( figure 3c ) . 

gratifyingly , invader probes with separated pseudocomplementary base 
 pairs and energetic hotspots display improved recognition efficiency 
 ( see curves for dsx1:dsx2 and dsx3:dsx4 , respectively , figure 3c ) , which follows the observed trend in grec293 values .
it is particularly noteworthy that 
 the use of as little as 1.0 mol equiv of dsx3:dsx4 results in 20% recognition of dh8 , especially 
 when considering that optimal invader design normally calls for incorporation 
 of multiple energetic hotspots .
this 
 suggests that spatial separation of pseudocomplementary base pairs 
 and energetic hotspots is a promising principle for the design of 
 efficient dsdna - targeting invader probes . in the present study , 

although there is an absence 
 of suitable pseudocomplementary base pairs for cg - steps , we have previously shown that energetic hotspots 
 can be comprised of intercalator - functionalized monomers that are 
 based on any of the natural nucleobases ( although there is a preference 
 for pyrimidine monomers ) .
it is , therefore , 
 likely possible to design dsx - like probes against dna 
 targets with a higher gc - content than studied herein .
recognition of dsdna 
 model target dh8 using different 
 invader probes . ( a ) illustration of recognition process ; ( b ) representative 
 electrophoretograms for recognition of dh8 using 1- to 
 500-fold excess of x5:x6 , dy5:dy6 dsx1:dsx2 , or dsx3:dsx4 ; ( c ) dose  response curves ( average of at least 
 three independent experiments , error bars represent standard deviation ) . 

the sequence of dna hairpin dh8 is shown in figure 4 . for experimental 
 conditions ,
lastly , binding specificity was 
 studied by incubating the invader 
 probes with dna hairpins dh9dh14 , which differ in the nucleotide sequence at one position in the 
 stem region relative to the probes ( figure 4a ) .
excellent discrimination of the nontarget 
 dna hairpins is observed , even when using a 200-fold molar excess 
 of x5:x6 , dy5:dy6 or dsx1:dsx2 ( figure 4b ) .
similarly , high - affinity invader probe dsx3:dsx4 only results in trace recognition of dh10 and dh14 .
to gain further insight into the 
 underlying reasons for the excellent binding fidelity , we evaluated 
 individual probe strands against singly mismatched single - stranded 
 dna targets ( tables s10s12 ) .
excellent 
 mismatch discrimination is generally observed . in agreement with the 
 observations from the dsdna - recognition experiments ,
the discrimination 
 is least efficient when dsx3 or dsx4 are 
 hybridized with single - stranded targets that have a sequence corresponding 
 to the target region in dh10 and dh14 ( tm s only reduced by 57 c , 
 relative to matched duplexes , table s12 ) .
the binding of invader probes to noncomplementary dsdna targets 
 would accordingly entail formation of two mismatched 
 and destabilized probe - target duplexes , which is energetically prohibitive 
 in most cases .
moreover , like other structured probes , the double - stranded 
 invader probes are also likely to exhibit improved binding specificity 
 due to a 
thus , pseudocomplementary invader probes allow for strong and highly 
 specific binding to mixed - sequence dsdna target regions at ionic conditions .
( a ) sequences and thermal denaturation temperatures of dna 
 hairpins with isosequential ( dh8 ) or nonisosequential 
 stems ( dh9dh14 ) ; underlined nucleotides 
 denote sequence deviations relative to invader probes .
( b ) representative 
 gel electrophoretograms illustrating incubation of dh8dh14 with 200-fold molar excess of x5:x6 , dy5:dy6 , dsx1:dsx2 , or dsx3:dsx4 . for experimental 
 conditions , see figure 2 .
a synthetic 
 route to 2-n-(pyren-1-yl)methyl-2-amino-2-deoxy-2-n - methyl-2-thiouridine phosphoramidite 6 has 
 been developed ( 10% overall yield over 13 steps from uridine ) . 

ons modified with the corresponding y monomer display 
 high affinity toward cdna ( tm up 
 to + 11.5 c ) .
incorporation of 2-aminoadenosine monomer d next to the y monomer further increases cdna 
 affinity .
i.e. , 5-yd-3:3-dy-5 cassettes ) are thermolabile and activated for 
 recognition of dsdna targets , but less so than regular invader probes 
 with hotspots comprised of 2-n-(pyren-1-yl)methyl-2-n - methyl-2-aminouridine monomer x. 
 in other words , close proximity of the two structural elements that 
 normally activate pseudocomplementary dna and invader probes for dsdna - recognition  i.e .
, 
 the weak base pairs between 2,6-diaminopurine and 2-thiouracil , and 
 the destabilizing + 1 interstrand zipper arrangements of intercalating 
 pyrene moieties  does not provide a clear design benefit .
in 
 contrast , dsx probes , in which the two destabilizing 
 structural motifs are separated , are strongly activated for recognition 
 of mixed - sequence dsdna targets .
this was confirmed in studies with 
 model dsdna targets as efficient recognition was demonstrated to occur 
 with excellent specificity .
thus , the use of chimeric pseudocomplementary 
 invader probes presents itself as a promising strategy for mixed - sequence 
 recognition of dsdna for applications in molecular biology and nucleic 
 acid diagnostics , especially since we have recently demonstrated that 
 conventional invader probes can recognize target regions in chromosomal 
 dna .
nucleoside 1(32 ) ( 3.40 g , 4.39 mmol ) was coevaporated 
 with anhydrous 1,2-dichloroethane ( 2  30 ml ) , redissolved in 
 anhydrous pyridine ( 55 ml ) and cooled to 0 c .
to this was added 
 4-dimethylaminopyridine ( dmap , 55 mg , 0.44 mmol ) and acetic anhydride 
 ( 1.25 ml , 13.18 mmol ) . after stirring at ambient temperature for 12 
 h
, the reaction mixture was diluted with etoac ( 150 ml ) and washed 
 with h2o ( 80 ml ) and saturated aqueous nahco3 ( 80 ml ) .
the organic layer was evaporated to near dryness and coevaporated 
 with absolute etoh : toluene ( 2:1 v / v , 3  30 ml ) . the resulting 
 residue ( 3.5 g )
was suspended in acoh : h2o ( 4:1 
 v / v , 55 ml ) and the reaction mixture was stirred overnight at room 
 temperature .
solvents were evaporated off and the resulting residue 
 was purified by silica gel column chromatography ( 05% meoh / ch2cl2 , v / v ) to afford o5-hydroxy derivative 
 ( 1.8 g ) as a white solid material .
this material was coevaporated 
 with anhydrous pyridine : ch2cl2 ( 1:1 v / v , 2  
 30 ml ) , redissolved in anhydrous pyridine : ch2cl2 ( 1:1 v / v , 44 ml ) and cooled to 20 c ( ice / salt ) .
methanesulfonyl 
 chloride ( 0.75 ml , 9.62 mmol ) was added dropwise over 30 min and the 
 reaction mixture was stirred at 20 c for 2 h more .
the 
 reaction mixture was diluted with ch2cl2 ( 80 
 ml ) and washed with saturated aqueous nahco3 ( 50 ml ) .
the 
 aqueous layer was back - extracted with ch2cl2 ( 3  15 ml ) and the organic layers were combined and evaporated 
 to dryness , followed by coevaporation with absolute etoh : toluene ( 2:1 
 v / v , 3  30 ml ) .
the resulting residue was purified by silica 
 gel column chromatography ( 03% meoh / ch2cl2 , v / v ) to afford nucleoside 2 ( 1.43 g , 55% ) as a white 
 foam .
rf = 0.4 ( 5% meoh 
 in ch2cl2 , v / v ) ; maldi - hrms m / z 614.1600 ( [ m + na ] , c30h29n3o8sna , calc . 

614.1568 ) ; h nmr ( 500 mhz , dmso - d6 )  11.47 ( d , 1h , ex , j = 2.2 hz , nh ) , 
 8.338.36 ( d , 1h , j = 9.0 hz , py ) , 8.258.29 
 ( ap t , 2h , j = 7.6 hz , py ) , 8.21 ( d , 1h , j = 7.5 hz , py ) , 8.15 ( br s , 2h , py ) , 8.108.12 ( d , 
 1h , j = 9.0 hz , py ) , 8.048.08 ( t , 1h , j = 7.6 hz , py ) , 7.92 ( d , 1h , j = 7.5 hz , 
 py ) , 7.73 ( d , 1h , j = 8.2 hz , h6 ) , 6.40 ( d , 1h , j = 7.4 hz , h1 ) , 5.67 ( dd , 1h , j = 8.2 hz , 2.2 hz , h5 ) , 5.39 ( dd , 1h , j = 6.6 hz , 
 3.6 hz , h3 ) , 4.444.48 ( m , 2h , h5 ) ,
4.344.43 
 ( m , 3h , ch2py , h4 ) , 3.84 ( ap t , 1h , j  7.5 hz , h2 ) , 3.20 ( s , 3h , ch3so2 ) , 2.34 ( s , 3h , ch3n ) , 2.13 ( s , 3h , ch3co ) ; c nmr ( 125 mhz , dmso - d6 )  169.7 , 162.7 , 150.5 , 140.6 ( c6 ) , 131.9 , 130.7 , 130.3 , 130.2 , 
 129.1 , 127.7 ( py ) , 127.3 ( py ) , 127.0 ( py ) , 126.8 ( py ) , 126.1 ( py ) , 
 125.1 ( py ) , 124.4 ( py ) , 124.2 , 123.8 , 123.7 ( py ) , 102.6 ( c5 ) , 83.7 
 ( c1 ) , 80.0 ( c4 ) , 71.7 ( c3 ) , 69.1 ( c5 ) , 
 64.9 ( c2 ) , 57.5 ( ch2py ) , 37.7 ( ch3n ) , 
 36.8 ( ch3so2 ) , 20.9 ( ch3co ) .
nucleoside 2 ( 0.77 g , 1.30 
 mmol ) was dried through coevaporation with anhydrous 1,2-dichloroethane 
 ( 3  6 ml ) and suspended in anhydrous etoh ( 25 ml ) .
to this was 
 added nahco3 ( 275 mg , 3.25 mmol ) and the reaction mixture 
 was refluxed for 4 days .
ch2cl2 ( 100 ml ) was 
 added and the precipitate was filtered off and washed with ch2cl2 .
the combined organic layers were evaporated 
 to dryness and the resulting residue was purified via silica gel column 
 chromatography ( 07% meoh in ch2cl2 , 
 v / v ) to afford nucleoside 3 ( 0.34 g , 52% ) as a white 
 foam .
rf = 0.3 ( 10% meoh 
 in ch2cl2 , v / v ) ; maldi - hrms m / z 522.1985 ( [ m + na ] , c29h29n3o5na , calc . 

522.1999 ) ; h nmr ( 500 mhz , dmso - d6 )  8.398.42 ( d , 1h , j = 9.3 
 hz , py ) , 8.248.28 ( m , 2h , py ) , 8.198.22 ( d , 1h , j = 8.0 hz , py ) , 8.14 ( br s , 2h , py ) , 8.038.10 ( d+t , 
 2h , py ) , 7.967.99 ( d , 1h , j = 8.0 hz , py ) , 
 7.93 ( d , 1h , j = 7.7 hz , h6 ) , 6.35 ( d , 1h , j = 8.5 hz , h1 ) , 5.83 ( d , 1h , j = 7.7 hz , h5 ) , 5.53 ( d , 1h , ex , j = 4.9 hz , 3oh ) , 
 5.12 ( t , 1h , ex , j = 5.2 hz , 5oh ) , 
 4.434.51 ( m , 3h , ch2py , h3 ) , 4.124.27 
 ( m , 2h , och2ch3 ) , 3.994.01 ( m , 1h , h4 ) , 3.583.62 
 ( m , 2h , h5 ) , 3.47 ( dd , 1h , j = 8.5 hz , 5.2 
 hz , h2 ) , 2.36 ( s , 3h , ch3n ) , 1.06 ( t , 3h , j = 7.1 hz ,
ch3ch2o ) ; c nmr ( 125 mhz , dmso - d6 )  169.3 , 155.0 , 138.1 ( c6 ) , 132.7 , 
 130.7 , 130.3 , 130.1 , 129.0 , 127.5 ( py ) , 127.3 ( py ) , 126.9 ( py ) , 126.8 
 ( py ) , 126.1 ( py ) , 125.02 ( py ) , 124.99 ( py ) , 124.4 ( py ) , 124.1 , 123.8 , 
 123.6 ( py ) , 108.4 ( c5 ) , 87.6 ( c4 ) , 85.0 ( c1 ) , 71.2 
 ( c3 ) , 68.8 ( c2 ) , 64.2 ( och2ch3 ) , 61.7 ( c5 ) , 
 57.1 ( ch2py ) , 39.0 ( ch3n  overlap with 
 dmso - d6 signal ) , 13.6 ( ch3ch2o ) .
nucleoside 3 ( 0.32 g , 0.63 
 mmol ) was coevaporated with anhydrous 1,2-dichloroethane ( 2  
 5 ml ) and redissolved in anhydrous pyridine ( 6 ml ) . to this
was added 
 dmtrcl ( 0.26 g , 0.76 mmol ) and 4-dimethylaminopyridine ( dmap , 8 mg , 
 0.06 mmol ) and the reaction mixture was stirred at ambient temperature 
 for 14 h at which point it was diluted with chcl3 ( 80 ml ) 
 and washed with saturated aqueous nahco3 ( 30 ml ) and h2o ( 30 ml ) .
the aqueous layer was back - extracted with chcl3 ( 3  15 ml ) and the combined organic layers were dried 
 ( na2so4 ) and evaporated to dryness .
the resulting 
 residue was purified by silica gel column chromatography ( 02.5% 
 meoh in chcl3 , v / v ) to afford nucleoside 4 ( 0.45 g , 88% ) as a pale orange foam .
rf = 0.7 ( 10% meoh in ch2cl2 , 
 v / v ) ; maldi - hrms m / z 824.3319 ( [ m 
 + na ] , c50h47n3o7na , calc .
824.3306 ) ; h nmr ( 500 
 mhz , dmso - d6 )  8.388.41 
 ( d , 1h , j
= 9.3 hz , py ) , 8.258.29 ( m , 2h , 
 py ) , 8.178.19 ( d , 1h , j = 7.8 hz , py ) , 8.118.15 
 ( 2d , 2h , j = 9.1 hz , 9.1 hz , py ) , 8.048.09 
 ( m , 2h , py ) , 7.988.00 ( d , j = 7.8 hz , py ) , 
 7.69 ( d , 1h , j = 7.8 hz , h6 ) ,
7.197.38 ( m , 
 9h , dmtr ) , 6.836.88 ( m , 4h , dmtr ) , 6.32 ( d , 1h , j = 7.9 hz , h1 ) , 5.605.64 ( m , 2d , 1 ex , j = 7.8 hz , 5.0 hz , h5 , 3oh ) , 4.484.54 ( m , 
 2h , ch2py ) , 4.444.48 ( m , 1h , h3 ) , 4.104.24 
 ( m , 3h , h4 , och2ch3 ) , 3.71 ( s , 3h , ch3o ) , 3.70 ( s , 3h , 
 ch3o ) , 3.523.58 ( dd , 1h , j = 7.9 
 hz , 5.8 hz , h2 ) , 3.313.35 ( dd , 1h , j = 10.5 hz , 5.0 hz , h5 - partial overlap with h2o ) , 3.153.20 ( dd , 1h , j = 10.5 hz , 3.5 hz , 
 h5 ) , 2.43 ( s , 3h , ch3n ) , 1.05 ( t , 3h , j = 7.0 hz ,
ch3ch2o ) ; c nmr ( 125 mhz , dmso - d6 )  169.2 , 158.09 , 158.08 , 154.9 , 144.5 , 
 138.1 ( c6 ) , 135.3 , 135.0 , 132.6 , 130.7 , 130.2 , 130.1 , 129.7 ( dmtr ) , 
 129.6 ( dmtr ) , 129.0 , 127.8 ( dmtr ) , 127.6 ( dmtr ) , 127.5 ( py ) , 127.3 
 ( py ) , 126.91 ( py ) , 126.86 ( py ) , 126.7 ( dmtr ) , 126.1 ( py ) , 125.0 ( py ) , 
 124.4 ( py ) , 124.1 , 123.9 , 123.6 ( py ) , 113.20 ( dmtr ) , 113.17 ( dmtr ) , 
 108.1 ( c5 ) , 85.9 , 85.7 ( c4 ) , 85.3 ( c1 ) , 71.0 ( c3 ) , 
 68.1 ( c2 ) , 64.2 ( och2ch3 ) , 64.0 ( c5 ) , 57.1 ( ch2py ) , 55.0 ( ch3o ) , 38.8 ( ch3n ) , 13.6 
 ( ch3ch2o ) .
a trace impurity of chcl3 was identified in 
 the c nmr at 79.1 ppm .
an ice - cold solution of anhydrous 1,1,3,3-tetramethylguanidine 
 ( tmg , 
 0.68 ml , 5.42 mmol ) in anhydrous pyridine ( 10 ml ) was saturated with 
 hydrogen sulfide gas for 1 h while maintaining the temperature at 
 0 c .
the solution was transferred , using an argon - flushed syringe , 
 to a precooled flask containing nucleoside 4 ( 0.44 g , 
 0.54 mmol ) and the reaction mixture was allowed to reach room temperature . 
 after stirring under an argon atmosphere for 72 h , etoac ( 100 ml ) 

was added and the organic layer was washed with saturated aqueous 
 nahco3 ( 50 ml ) and h2o ( 50 ml ) .
the aqueous 
 layer was back - extracted with ch2cl2 ( 3  
 20 ml ) and the combined organic layers were evaporated to dryness 
 and coevaporated with absolute etoh : toluene ( 2:1 v / v , 3  15 
 ml ) .
the resulting residue was purified by silica gel column chromatography 
 ( 070% etoac in petroleum ether , v / v ) to afford nucleoside 5 ( 0.35 g , 82% ) as a white foam .
rf = 0.8 ( 80% etoac in petroleum ether , v / v ) ; maldi - hrms m / z 812.2765 ( [ m + na ] , c48h43n3o6sna , calc .
812.2797 ) ; h nmr ( 500 mhz , dmso - d6 )  12.77 ( br s , 1h , ex , nh ) , 8.498.52 
 ( d , 1h , j = 9.3 hz , py ) , 8.248.29 ( m , 2h , 
 py ) , 8.178.20 ( d , 1h , j = 8.0 hz , py ) , 8.128.16 
 ( 2d , 2h , j = 9.1 hz , 9.1 hz , py ) , 8.028.11 
 ( m , 3h , py ) , 7.75 ( d , 1h , j = 8.2 hz , h6 ) , 7.207.39 
 ( m , 10h , dmtr , h1 ) , 6.846.89 ( m , 4h , dmtr ) , 5.62 ( dd , 
 1h , j = 8.2 hz , 1.7 hz , h5 ) , 5.55 ( d , 1h , ex , j = 4.9 hz , 3oh ) , 4.534.57 ( d , 1h , j = 13.0 hz ,
ch2py ) , 4.434.51 ( m , 2h , 
 ch2py , h3 ) , 4.084.12 ( m , 1h ,
h4 ) , 
 3.71 ( s , 3h , ch3o ) , 3.70 ( s , 3h , ch3o ) , 3.483.52 
 ( m , 1h , h2 ) , 3.333.37 ( dd , 1h , j = 
 10.5 hz , 5.0 hz , h5 ) , 3.183.22 ( dd , 1h , j = 10.5 hz , 3.0 hz , h5 ) , 2.44 ( s , 3h , ch3n ) ; c nmr ( 125 mhz , dmso - d6 )  
 176.6 , 159.0 , 158.11 , 158.10 , 144.5 , 140.8 ( c6 ) , 135.3 , 135.0 , 132.6 , 
 130.7 , 130.3 , 130.2 , 129.73 ( dmtr ) , 129.68 ( dmtr ) , 129.2 , 128.0 ( py ) , 
 127.9 ( dmtr ) , 127.6 ( dmtr ) , 127.3 ( py ) , 127.0 ( py ) , 126.8 ( py ) , 126.7 
 ( dmtr ) , 126.1 ( py ) , 125.02 ( py ) , 125.00 ( py ) , 124.4 ( py ) , 124.1 , 124.0 
 ( py ) , 123.9 , 113.3 ( dmtr ) , 113.2 ( dmtr ) , 106.9 ( c5 ) , 88.0 ( c1 ) , 
 86.0 , 85.4 ( c4 ) , 71.0 ( c3 ) , 68.6 ( c2 ) , 63.9 
 ( c5 ) , 57.6 ( ch2py ) , 55.0 ( ch3o ) , 39.2 
 ( ch3n ; overlap with dmso - d6 ) . to a flame - dried round - bottomed flask containing 
 nucleoside 5 ( 150 mg , 0.19 mmol ) was added anhydrous 
 ch2cl2 ( 2 ml ) , anhydrous n , n - diisopropylethylamine ( dipea , 165 l , 0.95 mmol ) 
 and 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite 
 ( pcl reagent , 85 l , 0.38 mmol ) .
the reaction mixture was stirred 
 at room temperature for 3.5 h at which point ice - cold etoh ( 1.0 ml ) 
 was added .
the reaction mixture was evaporated to dryness and the 
 resulting residue was purified by silica gel column chromatography 
 ( 055% etoac in petroleum ether , v / v ) followed by precipitation 
 from cold petroleum ether to afford nucleoside 6 ( 153 
 mg , 81% ) as a white foam . rf = 0.6 ( 50% etoac in petroleum ether , v / v ) ; maldi - hrms m / z 1012.3843 ( [ m + na ] , c57h60n5o7psna , calc .
1012.3887 ) ; p nmr ( 121 mhz , cdcl3 )  150.9 , 149.6 .
modified 
 ons were synthesized on a 0.2 mol scale using a dna synthesizer , 
 succinyl - linked lcaa - cpg ( long chain alkyl amine controlled pore glass ) 
 columns with a pore size of 500  , and standard protocols for 
 incorporation of a , c , g and 
 t dna phosphoramidites .
the following hand - coupling conditions were 
 used for incorporation of the corresponding phosphoramidites of monomers x , y , s ( n3/o4-toluoyl protected ) 
 and d ( bis(diisobutylaminomethylidene)-protected ) ( coupling 
 time ; activator ; coupling yield ) : x ( 15 min ; 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole ; 99% ) , y ( 15 min ; 4,5-dicyanoimidazole ; 
 95% ) and s / d ( 15 min ; 4,5-dicyanoimidazole ; 
 99% ) .
modified phosphoramidites were used at 50-fold molar 
 excess and 0.05 m concentration in ch3cn .
extended oxidation 
 ( 45 s ) with standard 0.05 m aqueous iodine was used for d1d4 and x1x6 .
extended oxidation ( 2  5 min oxidation with an acetonitrile 
 wash between oxidations ) using a tert - butylhydroperoxide / ch3cn / h2o solution ( 10/87/3 , v / v / v ) was used for all 
 ons containing s and y modifications to 
 prevent desulfurization .
cleavage from 
 solid support and removal of protecting groups was accomplished by 
 treatment with 32% aq .
ons were 
 purified in the dmt - on mode via ion - pair reverse phase hplc ( c18 column ) using a 0.05 m triethylammonium acetate 
. acoh ) and precipitation 
 ( naoac / naclo4/acetone , 18 c for 1216 
 h ) .
the identity of synthesized ons was established through maldi - ms 
 analysis ( table s1 ) recorded in positive 
 ions mode on a quadrupole time - of - flight tandem mass spectrometer 
 equipped with a maldi source using anthranilic acid , 3-hydroxypicolinic 
 acid ( 3-hpa ) or 2,4,6-trihydroxyacetophenone ( thap ) as matrices .
purity 
 was verified by ion - pair reverse phase hplc running in analytical 
 mode ( > 90% unless otherwise mentioned ) . on concentrations 

were estimated using the following extinction coefficients for dna 
 ( od/mol ) : g ( 12.01 ) , a ( 15.20 ) , t ( 8.40 ) , c ( 7.05 ) ; rna ( od/mol ) : 
 g ( 13.70 ) , a ( 15.40 ) , u ( 10.00 ) , c ( 9.00 ) ; pyrene ( 22.4),d ( 8.5 ) , s ( 10.0 ) 
 and y ( 32.4 ) .
strands were 
 thoroughly mixed and denatured by heating to 7085 c , 
 followed by cooling to the starting temperature of the experiment . 

thermal 
 denaturation temperatures ( tm s ) 
 of duplexes ( 1.0 m final concentration of each strand ) were 
 measured using a uv / vis spectrophotometer equipped with a 12-cell 
 peltier temperature controller and determined as the maximum of the 
 first derivative of thermal denaturation curves ( a260 vs t ) recorded in medium salt phosphate 
 buffer ( tm buffer : 100 mm nacl , 0.1 mm 
 edta and ph 7.0 adjusted with 10 mm na2hpo4 and 
 5 mm na2hpo4 ) .
the temperature of the denaturation 
 experiments ranged from at least 15 c below tm to 20 c above tm ( although 
 not below 3 c ) .
thermodynamic parameters for 
 duplex formation were determined through 
 fitting of baselines of denaturation curves ( vant hoff analysis ) 
 using software provided with the uv / vis spectrometer .
bimolecular 
 reactions , two - state melting behavior , and a heat capacity change 
 of cp = 0 upon hybridization were 
 assumed .
a minimum of two experimental 
 denaturation curves were each analyzed at least three times to minimize 
 errors arising from baseline choice .
uv  vis 
 absorption 
 spectra ( range 200600 nm ) were recorded at 10 c using 
 the same samples and instrumentation as in the thermal denaturation 
 experiments .
steady - state fluorescence emission spectra of y- or dy - modified ons and the corresponding duplexes 
 with complementary 
 dna / rna targets , were recorded in nondeoxygenated thermal denaturation 
 buffer ( each strand at 1.0 m concentration ) and obtained as 
 an average of five scans using an excitation wavelength of ex = 350 nm .
excitation and emission slits of 5.0 and 2.5 nm , 
 respectively , were used along with a scan speed of 600 nm / min .
experiments 
 were determined at 5 c ( to ascertain maximal hybridization of 
 probes to dna / rna targets ) under n2 flow ( to prevent condensation ) .
unmodified dna hairpins dh1dh14 were obtained from commercial sources and used without 
 further purification .
the dna hairpins were 3-dig - labeled 
 using the second generation dig gel shift kit ( roche applied bioscience ) 
 following the manufacturer s recommendation .
dig - labeled ons 
 obtained in this manner were diluted and used without further purification 
 in the recognition experiments .
preannealed probes ( 85 c for 
 10 min , cooled to room temperature over 15 min ) and dig - labeled dna 
 hairpins ( 34.4 nm ) were mixed and incubated in hepes buffer ( 50 mm 
 hepes , 100 mm nacl , 5 mm mgcl2 , 10% sucrose , 1.44 mm spermine 
 tetrahydrochloride , ph 7.2 ) for the specified time at ambient temperature 
 ( 21  3 c ) .
the reaction mixtures were then diluted 
 with 6x dna loading dye ( fermentas ) and loaded onto a 16% nondenaturing 
 polyacrylamide gel .
electrophoresis was performed using a constant 
 voltage of 70 v for 2.5 h at 4 c using 0.5 tbe 
 as a running buffer ( 45 mm tris , 45 mm boric acid , 1 mm edta ) .
gels 
 were blotted onto positively charged nylon membranes ( roche applied 
 bioscience ) using constant voltage with external cooling ( 100 v , 4 
 c ) .
the membranes were exposed to antidigoxigenin - ap fab fragments as recommended by the manufacturer of the dig gel shift 
 kit , transferred to a hybridization jacket , and incubated with the 
 substrate ( cspd ) in detection buffer for 10 min at 37 c .
the 
 chemiluminescence of the formed product was captured on x - ray film , 
 which was developed using an x - omatic 1000a x - ray film developer ( kodak ) . 

the efficiency 
 of dna recognition was determined as the intensity ratio between the 
 recognition complex band and the total lane .
nonlinear 
 regression was used to fit data points from dose  response experiments , 
 using a script written for the  solver  module in microsoft 
 office excel .
the following nomenclature 
 describes the relative arrangement between two pyrene - functionalized 
 monomers positioned on opposing strands in a duplex : the number n describes the distance measured in number of base pairs 
 and has a positive value if a monomer is shifted toward the 5-side 
 of its own strand relative to a second reference monomer on the other 
 strand .
conversely , n has a negative value if a monomer 
 is shifted toward the 3-side of its own strand relative to 
 a second reference monomer on the other strand .
 the development of molecular strategies 
 that enable recognition 
 of specific double - stranded dna ( dsdna ) regions has been a longstanding 
 goal as evidenced by the emergence of triplex - forming oligonucleotides , 
 peptide nucleic acids ( pnas ) , minor groove binding polyamides , and  
 more 
 recently  engineered proteins such as crispr / cas9 . despite this 
 progress , 
 an unmet need remains for simple hybridization - based probes 
 that recognize specific mixed - sequence dsdna regions under physiological 
 conditions . 
 herein , we introduce pseudocomplementary invader probes as a step in this direction . these double - stranded probes 
 are chimeras between pseudocomplementary dna ( pcdna ) and invader probes , 
 which are activated for mixed - sequence dsdna - recognition through the 
 introduction of pseudocomplementary base pairs comprised of 2-thiothymine 
 and 2,6-diaminopurine , and + 1 interstrand zipper arrangements of intercalator - functionalized 
 nucleotides , respectively . 
 we demonstrate that certain pseudocomplementary 
 invader probe designs result in very efficient and specific recognition 
 of model dsdna targets in buffers of high ionic strength . 
 these chimeric 
 probes , therefore , present themselves as a promising strategy for 
 mixed - sequence recognition of dsdna targets for applications in molecular 
 biology and nucleic acid diagnostics . 
Introduction
Results and Discussion
Conclusion
Experimental
Section