Patent Application: US-46071899-A

Abstract:
fluorescent energy transfer cassettes that allow through bond energy transfer and have a succinimidyl ester functionality suitable for affecting them to biomolecules or provided and are applied to high throughput dna sequencing .

Description:
the preferred embodiments of the present invention and its advantages are best understood by referring to the fig1 through 4 of the drawings according to the teachings of the invention . several conclusions may be made based on the discussions above . first , enhancement of fluorescence emission is desirable . second , both dye - primer and dye - terminator approaches are viable , and the selection of one over the other is not a clear cut choice . it is possible , for instance , that if the dye - terminator methodology were improved then most sequencing reactions might be done that way . if they were , the number of reactions necessary to generate dna complements would be reduced by a factor of four relative to dye - primer approaches ( since the four different ddntp &# 39 ; s can be mixed ), and pausing bands would become invisible . there are at least two ways to improve the utility of the dye - terminator approach . energy transfer emission enhanced fluorescent tags for ddntp &# 39 ; s have not yet been developed , so work in this area is very likely to be useful . another avenue to explore is to devise completely new fluorescent labels for ddntp &# 39 ; s . superior dye - primer labels must overcome the false priming and mobility shift problems , and the experimental inconveniences associated with label incorporation . when singly labeled dye primers are used , mobility differences are compensated by virtual corrections to the data after detection but prior to output of the read . almost all of the work with double dye primers involves labels supported on t - analogs which constitute part of the primer sequence . those systems can be vulnerable to false priming due to these unnatural nucleotides . moreover , the mobility correction varies with sequence and cannot always be adequately accommodated by virtual corrections . to address this problem , mathies and co - workers designed a system that they term “ cassette labeling ”. [ a16 ] in this method , the primer syntheses are performed in such a way that the first fluorescent base is added at the 5 ′- termini of the primers , then six more cycles of phosphoramidite couplings are performed using deoxyribose units with no purine or pyrimidine functionalities . finally , the other label is added at the end of this chain . the primers formed in this way are less vulnerable to false priming , and the dna complements derived from them have improved mobility characteristics and exhibit less fluorescence quenching . however , this strategy is not ideal for several reasons . most important of these are the fact that these primers require seven more coupling steps than are required to generate the primer sequence . the word “ cassette ” is inappropriate for this system because it implies that the dye labels with the appropriate spacing are simply slotted in ; in fact , they are built on the end of the primer in a multi - step operation which must be repeated for each primer . second , the linker between the two labels is flexible . consequently , the fluorescence emission will be the result of averaged conformational states which may vary according to the different environments of the label system . third , the radiation that can be used to excite the labels must be chosen within relatively restrictive wavelength regions ( e . g . 488 nrn or 154 nrn source , but not ones much lower in wavelength ) . an eight dye system with four responding to one excitation wavelength and four responding at another would be extremely hard or almost impossible to develop given the dyes available . finally , the issue of gel mobilities is not a solved problem because different conformational states may still be present in ratios that vary with the peripheral primer sequence . energy transfer systems based on bodipy dyes have been introduced for enhanced sensitivity and improved gel mobility factors in dna sequencing , [ a17 ] but the concerns outlined above still apply . [ a18 ] dye - primer methodologies may be improved by generating a double - dye cassette that could be conveniently incorporated into a primer in one step . this cassette is preferably relatively rigid to minimize sequence dependent mobility variations . no compounds of the this type ( i . e ., fluorescent compounds having a uv - harvesting group in conjugation with a fluorescence emitter ) have been reported by others specifically for dna sequencing or , as far as we are aware , for other applications in biotechnology . however , compounds which harvest uv radiation , and transmit it to a fluorescent group via a conjugated system have been reported . this section summarizes highlights from that literature . polymers and oligormers of type i are prepared via sonogashira couplings of aryl halide and arylalkyne components . this coupling reaction is more efficient than the wadsworth - emmons reaction generally used to produce the corresponding systems with alkene rather than aryne linkages . in fact , solid phase syntheses of these materials are possible as a direct consequence of the efficiency of the sonogashira coupling . extended aryl alkyne molecules are not particularly soluble in any common solvent , but the alkoxide subsituents shown in structure i can be used to give appreciable solubilities . in nearly all literature on these compounds the “ or ” functionalities are o - hydrocarbons included for compatibility with organic media , although in at least one case a water soluble system has been produced when r was a sulfonated benzylic group . molecules of type i are chemically robust . they would not , for instance , react or decompose under the thermal cycling conditions used for enzymatic generation of dna components . the spacing between repeat units in the polymers i is approximately 6 . 75 å . this rigidity could be exploited to hold a uv absorbing group and a fluorescence emitter at a relatively invariant and easily estimated separation . photophysical properties of framework i are such that absorption occurs at around 448 nm and fluorescence emission occurs at 474 nm ( n =˜ 22 ). high fluorescent quantum yields are often observed ( ca 0 . 8 to 0 . 9 for many molecules of type i ) presumably because the rigidity of the system precludes bond motions that would otherwise result in radiationless decay . moreover , the emission spectrum tends to be relatively sharp , much sharper than the absorption spectrum . the most relevant property of these materials to this project is the energy transfer properties seen for molecules like polymer ii . this material emits at 524 nm irrespective of the wavelength of the absorption . it appears that random excitation of the polymer backbond transmits the energy to the low energy anthracene end group resulting in emission . 25 . the mechanism of energy transfer is “ photonic ”, rather than through space , hence the rigid arylalkyne rods have been referred to as “ molecular wires ”. the florescence quantum yield is not always high , however . if the low energy group is situated in a relatively central position in the polymeric chain , for instance , then the florescence quantum yield is low . however , placement of a high energy absorbing group at one termini and a low energy emitting group at the other facilitates tunable absorption and photonic relay to the low energy emitting group with high florescence quantum yield . thus , a particular wavelength of the absorption can be determined by choosing a suitable high energy end - group , and the wavelength of emission can be similarly adjusted using a different group at the other terminus . these types of observations regarding the photophysical properties of repeating arylalkyne units have been reported for many ohgomeric . cyclic , dendric , and polymeric systems . particularly relevant are systems which incorporate the so - called bodipy dyes , prepared for goals that were not stated to include dna sequencing . in conclusion , dye - primer methodologies could be improved by generating a double - dye cassette that could be conveniently incorporated into a primer in one step . this cassette should be relatively rigid to minimize sequence dependent mobility variations . the state of art in fluorescence - based dna sequencing methodologies is to use four sets of two dyes , one set for each sequencing reaction , arranged such that through space fluorescence energy transfer ( fet ) enhances the emission of each set . [ b1 - b4 ] this leads to increased sensitivity hence more bases can be sequenced in each run . typically , identical donor dyes are applied in each set so that irradiation at a single wavelength , usually 488 nm from argon lasers , can be used throughout . in a system wherein four dye sets are excited by one laser source , fet allows the dyes with longer fluorescence emission max values to absorb energy at 488 nm more efficiently . fet essentially increases the overlap of the absorption spectra of the acceptor dye with the exciting irradiation . fig1 a and 1 b are schematic diagrams illustrating energy transfer “ through space ” and energy transfer “ through bonds ,” respectively , for the production of fluorescent labels for biological systems . the first attempts to construct fet - based dye systems for dna sequencing used donor and acceptor dyes attached to different nucleobases in a primer . this route to fet - based dye systems is experimentally inefficient if many dye - labeled primers must be produced , as in the human genome project . moreover , the only mechanism by which these dyes could transfer energy would be “ through space .” ( fig2 ). several groups have noted that fet between two dyes that are conjugated with each other can be remarkably efficient , and this property has been used to produce new materials with interesting photophysical properties . [ b5 - b9 ] however , to the best of our knowledge , such “ through bond ” energy transfer ( fig1 b ) has not yet been exploited in the production of fluorescent labels for biological systems . disclosed here are our preliminary efforts to form fet - dye cassettes for biological labeling that potentially allow energy to be transferred through bonds as well as through space . specifically , the targets of this preliminary study are compounds 1aa , 1ab , 2aa , and 2ab . the issues to be addressed here are the syntheses of these compounds and , for 1ab , and 2ab , correlation of fluorescence properties related to orientation of the donor and acceptor fragments a and b , respectively . fig2 is a diagram illustrating the structures of four cassettes used according to the teachings of the present invention for labeling dna or other biological molecules , and fig3 is a diagram illustrating synthesis of cassettes 1 and 2 . briefly , the cassettes 1aa , 1ab , 2aa , and 2ab were constructed as outlined in fig2 . the bodipy [ b10 ] framework of compounds 3 was prepared by condensing 4 - iodobenzoyl chloride with the corresponding pyrrole , then reaction with bf 3 . oet 2 in a one - pot two - step process . [ b11 ] building blocks 4 were prepared from this produce via a gonogashira coupling reaction [ b12 ] with trimethylsilylethyne , then desilylation using tetra - n - butylammonium fluoride ( tabf ). bodipy &# 39 ; s 4a and b were then coupled to the core fragments 5 and 6 in the stepwise reactions indicted . these selective sonogashira couplings exploited the difference in reactivity between aryl iodides and aryl bromides . [ b13 , b14 ] fig4 summarizes important spectroscopic data for the cassettes 1 and 2 . the absorption spectra of the cassettes resemble the sum of the two individual chromophores , and the max values are not shifted relative to their bodipy constituents 4a / 4b . similarly , the fluorescence emission max values are not shifted relative to the corresponding acceptor fragment ( 4a or 4b ) alone . the emission of the donor fragment 4a , however , is almost completely suppressed in the cassettes 1ab and 2ab implying that the energy transfer efficiency in these systems is very high . possibly the most important data set in table 1 is the ratio of fluorescence intensities when the cassettes are irradiated at 488 nm . the relative increase in fluorescence intensity is greatest for the meta - substituted systems 1ab showing that this arrangement of donor and acceptor fragments is preferred over the para - orientation in cassette 2ab . two embodiments of the invention are depicted below . these two dyes a and b have absorption and fluorescence emission spectra that are comparable with the concept designed herein . one has a functional group that allows the tag to be attached to an amino group of a modified dna residue . dyes according to the invention may be used in sequencing reactions . for example , they may be attached to dna oligomers in connection with the “ dye - primer ” sequencing method . dideoxynucleoside triphosphate terminators may be tagged with the new dye systems in the “ dye terminator ” approach . ideal combinations of dyes may be developed as desired , as well as optimizing water solubility and minimizing gel mobility shifts between the dye systems . in the practice of the invention a multitude of uv absorbing and fluorescent emitting groups may be useful . suitable uv absorbers may include , but are not restricted to , perylene , anthacene , tetracene , fluorescein , and some bodipy dyes . suitable emitters may include , but are not restricted to , fluorescein derivatives , rhodamine systems , bodipys , squareine and other relatively long wavelength emitters such as cyanine dyes . energy transfer through space may be used in some members of the dye sets for convenience . additional embodiments of the invention are described in the paper attached as appendix a . experiments are in progress to further understand the fluorescence properties of these molecules ; determination of the relative contributions of through - space and through - bond energy transfer would be particularly interesting . others have speculated that through space fet can dominate even in conjugated systems . [ b15 , b16 ] and a significant contribution via this mechanism could account for the higher fluorescence emission of the meta - cassette 1ab . in general , the type of cassettes introduced in this communication could have several attractive features . specifically , through bond effects could add to the fet efficiency , the range of accessible max values may be greater due to through - bond energy transfer ( an important factor in four - color dan sequencing methodologies ), and the donor and acceptor fragments are packaged in a single facilitating convenient introduction of the tag . information related to experiments resulting in the above - described results is described below . characterization data for the cassettes . 1aa . mp 180 180 ° c . ( dec . ); r f 0 . 39 ( 45 % etoac / hexanes ); 1 h nmr ( cdcl 3 , 300 mhz ) δ1 . 41 ( s , 12 h ), 2 . 54 ( s , 12 h ), 2 . 91 ( bs , 4 h ), 5 . 98 ( s , 4 h ), 7 . 30 ( d , j = 8 . 4 hz , 4 h ), 7 . 67 ( d , j = 8 . 4 hz , 4 h ), 7 . 99 ( t , j = 1 . 5 hz , 1 h ), 8 . 26 ( d , j = 1 . 5 hz , 2 h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ14 . 6 , 25 . 6 , 88 . 0 , 91 . 0 , 121 . 4 , 123 . 1 , 124 . 6 , 126 . 2 , 128 . 4 , 131 . 1 , 132 . 4 , 132 . 9 , 135 . 7 , 139 . 8 , 140 . 4 , 142 . 9 , 155 . 8 , 160 . 6 , 168 . 9 ; ms ( fab *) m / z 911 ( m *); hrms calcd for c 53 h 43 n 5 o 4 b 2 f 4 [ m *] 911 . 3454 , found [ m *] 911 . 3460 . 1ab . mp 220 ° c . ( dec . ); r f 0 . 39 ( 35 % etoac / hexanes ); 1 h nmr ( cdcl 3 , 300 mhz ) δ0 . 97 ( t , j = 7 . 5 hz , 6 h ), 1 . 31 ( s , 6 h ), 1 . 42 ( s , 6 h ), 2 . 52 ( s , 6 h ), 2 . 54 ( s , 6 h ), 2 . 90 ( q , j = 7 . 3 hz , 4 h ), 2 . 92 ( bs , 4 h ), 5 . 99 ( s , 2 h ), 7 . 31 ( d , j = 8 . 1 hz , 4 h ), 7 . 66 ( d , j = 8 . 4 hz , 2 h ), 7 . 67 ( d , j = 8 . 4 hz , 2 h ), 7 . 99 ( t , j = 1 . 5 hz , 1 h ), 8 . 26 ( m , 2 h ), 13 c nmr ( cdcl 3 , 75 mhz ) δ11 . 9 , 12 . 5 , 14 . 6 , 25 . 6 , 87 . 9 , 88 . 0 , 91 . 0 , 91 . 2 , 121 . 4 , 122 . 8 , 123 . 1 , 124 . 5 , 124 . 6 , 126 . 2 , 128 . 4 , 128 . 7 , 130 . 4 , 131 . 1 , 132 . 3 , 132 . 4 , 133 . 0 , 135 . 7 , 136 . 6 , 138 . 1 , 138 . 9 , 139 . 8 , 140 . 4 , 142 . 9 , 154 . 1 , 155 . 8 , 160 . 7 , 168 . 9 ; ms ( fab +) m / z 967 ( m *); hrms calcd for c 57 h 51 n 5 o 4 b 2 f 4 [ m +] 967 . 4081 , found [ m +] 967 . 4101 . 2aa . mp 219 - 220 ° c . ; r f 0 . 32 ( 40 % etoac / hexanes ); 1 h nmr ( cdcl 3 , 300 mhz ) δ1 . 42 ( s , 12 h ), 2 . 55 ( s , 12 h ), 2 . 95 ( bs , 4 h ), 5 . 99 ( s , 4 h ), 7 . 28 - 7 . 33 ( m , 4 h ), 7 . 66 - 7 . 79 ( m , 6 h ), 8 . 37 ( d , j = 1 . 7 , 1 h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ14 . 57 , 14 . 6 , 25 . 8 , 88 . 1 , 88 . 8 , 92 . 2 , 97 . 3 , 121 . 4 , 123 . 3 , 123 . 4 , 123 . 6 , 124 . 6 , 128 . 3 , 128 . 5 , 129 . 8 , 131 . 2 , 132 . 4 , 132 . 7 , 134 . 3 , 134 . 4 , 135 . 9 , 136 . 0 , 142 . 9 , 155 . 9 , 160 . 3 , 169 . 0 ; ms ( fab +) m / z 911 ( m +); hrms calcd for c 53 h 43 n 5 o 4 b 2 f 4 [ m +] 611 . 3454 , found [ m *′ 911 . 3460 . 2ab . mp 215 - 216 ° c . ; r f 0 . 32 ( 40 % etoac / hexanes ); 1 h nmr ( cdcl 3 , 300 mhz ) δ0 . 96 ( t , j = 7 . 5 hz , 6 h ), 1 . 31 ( s , 6 h ), 1 . 40 ( s , 6 h ), 2 . 28 ( q , j = 7 . 3 hz , 4 h ), 2 . 51 ( s , 6 h ), 2 . 54 ( s , 6 h ), 2 . 94 ( bs , 4 h ), 5 . 97 ( s , 2 h ), 7 . 27 - 7 . 32 ( m , 4 h ), 7 . 68 - 7 . 73 ( m , 6 h ), 8 . 38 ( d , j = 1 . 5 hz , 1 h ); 13 c nmr ( cdcl 3 , 75 mhz ) δ11 . 9 , 12 . 5 , 14 . 6 , 14 . 7 , 17 . 1 , 25 . 7 , 29 . 7 , 88 . 1 , 88 . 6 , 92 . 3 , 97 . 3 , 121 . 4 , 123 . 0 , 123 . 4 , 123 . 5 , 124 . 6 , 125 . 3 , 126 . 8 , 128 . 2 , 128 . 3 , 128 . 7 , 129 . 0 , 130 . 5 , 131 . 1 , 132 . 3 , 132 . 7 , 133 . 0 , 134 . 35 , 134 . 4 , 135 . 8 , 136 . 0 , 136 . 6 , 138 . 2 , 139 . 0 , 141 . 1 , 143 . 0 , 154 . 1 , 155 . 8 , 159 . 1 , 160 . 2 , 169 . 1 ; ms ( fab +) m / z 967 ( m +); hrms calcd for c 57 h 51 n 5 o 4 b 2 f 4 [ m +] 967 , 4081 , found [ m +] 967 . 4101 . although the present invention and its advantage have been described in detail , it should be understood that various changes , substitutions , and alternatives can be made therein without departing from the spirit and scope of the present invention as defined by the appended claims . a1 . a rapid method for determining sequences in dna by primed synthesis with dna polymerase , f . sanger , and a . r . coulson , j . mol biol ., 1975 , 94 , 441 - 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7 . [**] a patent application entitled , “ through bond energy transfer in fluorescent dyes for labeling biological molecules ” has been filed . we thank dr c . i . martinez ( baylor college of medicine ) for valuable discussions . financial support for this work was provided by the nih ( hg0 1745 ) and the robert a . welch foundation . a . b . thanks the deutsche forschungsgemeinschaft for a fellowship . fluorescent dyes are central to many applications in biotechnology . for instance , all of the methods being used for sequencing the human genome involve fluorescence detection ,[ 1 ] as do the current technologies for visualizing dna arrays for genomics . [ 2 ] however , the dyes that are currently available are far from perfect . the important case of automated dna sequencing illustrates the type of limitation that is encountered . as dna fragments emerge from chromatographic separation they are irradiated by a single laser source and the fluorescence of the dye - label is monitored . two characteristics of the fluorescence emission properties are critical for such four - color sequencing schemes : resolution of λ max emis values for the dyes , and their fluorescence intensities . these two requirements place opposing constraints on the system . resolution requires emissions at wavelengths that are distinct from each other and from that of the excitation source . for single dye systems , fluorescence intensities are inversely related to their λ max emis values . this is because longer wavelength fluorescence emissions correlate with longer wavelength absorption maxima that overlap less with the laser source . through - space energy transfer dyes were developed to alleviate this problem , but still they require overlap of the donor emission with the acceptor absorption and this places constraints on the system . consequently , the dye sets currently used in dna sequencing , including the state - of - the - art through - space energy transfer systems [ 3 ] introduced in the last five years , [ 4 - 10 ] represent a compromise between overall resolution and intensity of the labels emitting at the longer wavelengths , as illustrated in fig7 a . this contrasts sharply with fig7 b , a representation of the type of spectroscopic performance that is sought after in this field . data presented in this paper illustrate that resolution / sensitivity issues are much easier to reconcile in dyes that exploit through - bond ( rather than through - space ) energy transfer . through - bond energy transfer has been widely investigated in the context of new materials [ 11 ] and models for photosynthesis , [ 12 - 16 ] but , to the best of our knowledge , this paper is the first to introduce the concept of using through - bond energy transfer to produce dyes for biotechnological applications . commercial dyes for dna sequencing are designed to collect light from an argon laser emitting at 488 or 514 nm . breaking with this paradigm , our dye set 1a - e incorporates a light - harvesting group that absorbs irradiation at 266 nm corresponding to lasers that operate on frequency doubling techniques . [ 17 ] use of such a laser source that emits at a relatively short wavelength , increases the λ - range available for detecting well - resolved fluorescence emissions . anthracene derivatives were chosen as the donor for the following reasons . anthracene has a strong uv absorption maximum at 250 nm ( ε = 80 , 000 m − 1 cm − 1 , in hexane ), [ 18 ] and substituted anthracenes tend to have slightly higher λ max abs . values . consequently , this simple aromatic system has good spectroscopic properties to capture light at 266 nm . anthracene derivatives are also cheap , easy to modify , and stable . 4 , 4 - difluoro - 4 - bora - 3a , 4a - diaza - s - indacene ( bodipy ®) units [ 19 ] were selected for the acceptor portion of dyes set 1 . these heterocycles tend to have strong , sharp fluorescence emissions , and they are compatible with sequencing methodologies ( aqueous environments , thermocycling etc ). [ 8 ] syntheses of the requisite aryl iodide units 2a - c were achieved via routine modifications of known procedures . [ 20 ] preparations of the compounds 2d and 2e will be reported separately . couplings of the donor ( anthracene - derivative ) and acceptor ( bodipy ) fragments were only notable insofar as the procedures used were easy to perform . they exploit the fact that aryl iodides are more reactive than aryl bromides in sonogashira couplings , hence selective reactions are possible ( fig9 a - 9 d ). [ 21 ] some spectroscopic properties of the dyes 1a - e are shown in table 1 . uv absorption spectra for these compounds represent an almost perfect superimposition of the donor and acceptor components . however , only the fluorescence emission peak of the acceptor bodipy fragment was observed for each of the dyes when they were excited at 270 nm ; quantitatively , the energy transfer efficiency for all these systems is over 95 %. [ 22 ] the fluorescence emission spectrum of anthracene has no significant overlap with the absorption spectrum of any of the bodipy acceptors , therefore this energy transfer must be predominantly via a through - bond mechanism . [ 23 ] consequently , the apparent stokes &# 39 ; shifts for the dyes are far greater than observed in any through - space energy transfer system . finally , the relative quantum yields [ 23 ] of these systems were all very similar . the mechanism of energy transfer is therefore likely to be similar for all the dyes in this series . electrochemical data were also collected for compounds 1a - e . briefly , two step reduction and oxidation processes were observed indicative of the anthracene and bodipy fragments behaving as independent entities . for instance , all five dyes showed a partially reversible oxidation between 0 . 92 and 0 . 97 v ( vs ferrocene in ch 2 cl 2 ) and the oxidation potential for 9 - bromo - 10 - ethynylanthracene 3 under the same conditions was measured as 0 . 95 v . overall , these spectroscopic and electrochemical data indicate that the components of dyes 1a - e behave as two distinct conjugated systems . however , energy transfer between the donor and acceptor fragments occurs with minimal fluorescence leakage from the donor . the λ max emis . values for these dyes span a 161 nm range ; this is approximately twice the corresponding { 80 nm } range observed for the classical dyes used for dna sequencing ( fam , joe , tamra , rox ). [ 4 ] fig8 a - d shows superimposed fluorescence spectra to illustrate the resolution involved . the relative intensities of dyes 1a - e ( 0 . 81 : 1 . 00 : 0 . 05 : 0 . 23 : 0 . 20 ) correspond to the relative intensities of the acceptor fragments . apparently , unlike through - space energy transfer dyes , their intensities do not diminish with their apparent stokes &# 39 ; shifts . the dyes described here are prototypes for labels that could be used in automated dna sequencing , but several obvious modifications need to be made to produce viable systems . in absolute terms , the intensities of these dyes are less than desired . further experimentation is in progress to identify through - bond energy transfer dyes composed of other donor / acceptor combinations to give improved resolution and fluorescence intensities . other desirable changes include ones to increase the water solubilities of these systems , and to standardize their gel mobilities for accurate base - calling . moreover , a facile method for attachment of the dyes to dna must be developed . in systems 1 the bromine functionality on the anthracene units is present to allow attachment of the dyes to dna ; thus , compound 1a has been transformed into the acid 4 , and coupling of this to dna is being investigated . these issues are , however , peripheral to the fundamental conclusion of this paper : through - bond energy transfer dyes have the potential to overcome problems that cannot be adequately addressed using single dye systems or through - space energy transfer cassettes . d relative quantum yield = ( observed fluorescence intensity of 1 when irradiated at 270 nm / observed fluorescence intensity of 1 when irradiated at absorption maxima of the acceptor ) × ( extinction coefficient of 1 at λ max abs . of acceptor / extinction coefficient of 1 of the donor at 270 nm ). [ 23 ] donor - acceptor fluorescent dye systems like 1 capture light from a blue laser and emit at much longer wavelengths . huge apparent stokes &# 39 ; shifts are observed and the dispersion of fluorescence emission maxima that can be observed for a single laser source is correspondingly large . through - bond energy transfer dyes such as this can give well - resolved and intensely fluorescent labels for dna sequencing and other applications . l . m . smith , j . z . sanders , r . j . kaiser , p . hughes , c . dodd , c . r . connell , c . heiner , s . b . kent , l . e . hood , nature 1986 , 321 , 674 - 9 . r . drmanac , i . labat , i . brukner , r . crkvenjakov , genomics 1989 , 4 , 114 - 28 . throughout “ donor groups ” are those which collect the irradiation , and “ acceptor groups ” are those which harvest it from the donor and emit . the terms “ through - space ” and “ through - bond ” refer to the mechanisms by which energy is transferred from the donor to the acceptor . j . ju , c . ruan , c . w . fuller , a . n . glazer , r . a . mathies , proc . natl . acad . sci . usa 1995 , 92 , 4347 - 51 . s . menchen , l . lee , p . thelsen , s . benson , k . upadhya , t . constantinescu , r . graham , s . spurgeon , b . rosenblum , s . koepf , r . o &# 39 ; neill , pe applied biosystems , technical report 1996 , 1 - 4 . j . ju , a . n . glazer , r . a . mathies , nature med . 1996 , 2 , 246 - 9 . j . ju , i . kheterpal , j . r . scherer , c . ruan , c . w . fuller , a . n . glazer , r . a . mathies , analytical biochem . 1995 , 231 , 131 - 40 . m . l . metzker , j . lu , r . a . gibbs , science 1996 , 271 , 1420 - 2 . l . g . lee , s . l . spurgeon , c . r . heiner , s . c . benson , b . b . rosenblum , s . m . menchen , r . j . graham , a . constantinescu , k . g . upadhya , j . m . cassel , nucleic acids research 1997 , 25 , 2816 - 22 . b . b . rosenblum , l . g . lee , s . l . spurgeon , s . h . khan , s . m . menchen , c . r . heiner , s . m . chen , nucleic acids research 1997 , 25 , 4500 - 4 . r . w . wagner , j . s . lindsey , pure & amp ; appl . chem . 1996 , 68 , 1373 - 80 . m . s . vollmer , f . würthner , f . effenberger , p . emele , d . u . meyer , t . stümpfig , h . port , h . c . wolf , chem . eur . j . 1998 , 4 , 260 - 9 . m . s . vollmer , f . effenberger , t . stümpfig , a . hartschuh , h . port , h . c . wolf , j . org . chem . 1998 , 63 , 5080 - 7 . f . li , s . i . yang , y . ciringh , j . seth , c . h . martin , d . l . singh , d . kim , r . r . birge , d . f . bocian , d . holten , j . s . lindsey , j . am . chem . soc . 1998 , 120 , 10001 - 17 . r . w . wagner , j . seth , s . i . yang , d . kim , d . f . bocian , d . holten , j . s . lindsey , j . org . chem . 1998 , 63 , 5042 - 9 . k . lauer , m . horio , ber . dtsch . chem . ges . 1936 , 69 , 130 - 137 . a . treibs , f .- h . kreuzer , liebigs ann . chem . 1968 , 718 , 208 - 23 . l . h . thoresen , h . kim , m . b . welch , a . burghart , k . burgess , synlett 1998 , 1276 - 8 . m . b . goldfinger , k . b . crawford , t . m . swager , j . am . chem . soc . 1997 , 119 , 4578 - 4593 . j .- l . mergny , t . garestier , m . rougée , a . v . lebedev , a . v . chassignol , n . t . thuong , c . héléne , biochemistry 1994 , 33 , 15321 - 8 . s .- i . kawahara , t . uchimaru , s . murata , chem . commun . 1999 , 8 , 563 - 4 . throughout “ donor groups ” are those which collect the irradiation , and “ acceptor groups ” are those which harvest it from the donor and emit . the terms “ through - space ” and “ through - bond ” refer to the mechanisms by which energy is transferred from the donor to the acceptor .