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WO2003080861A1 - Sequençage de molecule unique au moyen de nucleotides marques par des phosphates - Google Patents

Sequençage de molecule unique au moyen de nucleotides marques par des phosphates Download PDF

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WO2003080861A1
WO2003080861A1 PCT/EP2003/002982 EP0302982W WO03080861A1 WO 2003080861 A1 WO2003080861 A1 WO 2003080861A1 EP 0302982 W EP0302982 W EP 0302982W WO 03080861 A1 WO03080861 A1 WO 03080861A1
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fluorescence
labeled
process according
nucleic acid
fluorescence dye
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Claus Seidel
Hans-Joachim Fritz
Christian Griesinger
Natalia N. Gaiko
Sylvia Berger
Joachim Fries
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Max Planck Gesellschaft zur Foerderung der Wissenschaften
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a process for sequencing nucleic acids wherein the nucleic acid molecule to be sequenced is sequentially degraded in the presence of a fluorescence labeled reagent wherein a fluorescence labeled nucleoside is formed having nucleobase-specific fluorescence ⁇ h-aracteristics.
  • the method is a single molecule sequencing procedure comprising a spatially resolved detection step, e.g. confocal detection.
  • the method comprises a confocal detection step.
  • novel nucleobase-specific fluorescence dyes and reagents containing said dyes are provided.
  • the object underlying the present invention was to provide a method wherein the disadvantages of previous single molecule sequencing protocols are avoided.
  • nucleic acid molecule to be sequenced need not be labeled previously.
  • the label is introduced during the sequential degradation wherein labeled monomeric degradation products, e.g. nucleoside diphosphates or triphosphates, particularly deoxyribonucleoside triphosphates (dNTPs) are released during a degradation step.
  • labeled monomeric degradation products e.g. nucleoside diphosphates or triphosphates, particularly deoxyribonucleoside triphosphates (dNTPs) are released during a degradation step.
  • nucleobase-specific labels e.g. fluorescence labels a determination of the nucleic acid sequence may be carried out.
  • a nucleic acid elongation reaction comprises providing a starting nucleic acid molecule (A) and a nucleotide monomer, oligomer or polymer (B) optionally in the presence of a catalyst (Cat) . Reaction of (A) and (B) results in the formation of an elongated product nucleic acid molecule (C) and a phosphate or pyrophosphate group (D) .
  • the basic concept of the invention relies on the principle that the equilibrium of this reaction, which facilitates the formation of energetically more favourable product molecules (C) and (D), can be influenced by addition of a large excess of the product molecule (D) in such a way, that the product molecule (C) is completely reverted into the educt molecule (A) .
  • a particular advantage of the present invention is the coupling of the backward reaction with the introduction of a label, e.g. a fluorescent label in the formed educt molecule (B) .
  • a label e.g. a fluorescent label in the formed educt molecule (B) .
  • a specific application of the process described above comprises the sequencing of DNA, wherein the catalyst is an enzyme with one of the following properties:
  • a template-dependent polymerase having pyrophosphorolytic activity such as a DNA- or RNA-polymerase or a reverse transcriptase
  • a template-independent enzyme having pyrophosphorolytic activity such as a terminal transferase, e.g. Terminal Deoxynucleotidyltransferase (TdT)
  • TdT Terminal Deoxynucleotidyltransferase
  • III a template-independent enzyme having phosphorolytic activity such as a polynucleotide phosphorylase.
  • the labeled reagent is selected from pyrophosphates or pyrophosphate analogs which carry a labeling group and which are capable of taking part in the degradation reaction. If the reaction is a phosphorolytic reaction, the labeled reagent is preferably a labeled phosphate or phosphate analog, which is capable of taking part in the degradation reaction. In a preferred embodiment an approach is provided that incorporates the fluorescent dye during the enzyme catalyzed sequential degradation of the DNA into dNTP ' s ( Figure 1 ) .
  • TdT Deoxynucleotidyltransferase
  • the reverse reaction the pyrophosphorolysis of the DNA using pyrophosphate as substrate - the 3 '-terminal dNMP is cleaved and a dNTP is formed.
  • TdT to catalyze the reverse reaction allows us to utilize this reaction for the sequential degradation of DNA while incorporating fluorescent dyes via the pyrophosphorolysis.
  • fluorescent dye labeled pyrophosphate is used instead of inorganic pyrophosphate in the pyrophosphorolysis reaction
  • the cleaved 3 '-terminal nucleotide forms a deoxynucleotide 5 '-triphosphate that carries the fluorescent dye at the y-phosphate.
  • a first aspect of the present invention relates to a process for sequencing of nucleic acids comprising:
  • a reagent labeled with a dye e.g. a fluorescence dye wherein said dye, e.g. fluorescence dye is capable to distinguish between nucleobases when bound thereto
  • a dye e.g. a fluorescence dye
  • said dye e.g. fluorescence dye is capable to distinguish between nucleobases when bound thereto
  • determining the nucleic acid sequence by sequential measurement of nucleobase-specific signals, e.g. fluorescence signals from the monomeric degradation products formed in (c) .
  • the degradation of the nucleic acid molecule of the invention comprises an enzymatic reaction, more preferably an enzymatic reaction wherein a sequential release of monomeric degradation products such as nucleotides occurs and wherein a labeled reagent, e.g. a labeled phosphate or pyrophosphate, or at least a labeled part thereof is incorporated into the liberated degradation product, e.g. a nucleoside diphosphate or triphosphate.
  • a labeled reagent e.g. a labeled phosphate or pyrophosphate, or at least a labeled part thereof is incorporated into the liberated degradation product, e.g. a nucleoside diphosphate or triphosphate.
  • the reagent is a pyrophosphate and the sequential degradation is catalyzed by an enzyme having pyrophosphorolytic activity such as Terminal Deoxynucleotidyltransferase (TdT), which is a commercially available enzyme.
  • TdT Terminal Deoxynucleotidyltransferase
  • the cleavage of the nucleic acid to be sequenced in the presence of pyrophosphate preferably results in the formation of y-P labeled nucleoside triphosphates.
  • an enzyme was used as a catalyst, which has the selectivity and property of an exonuclease, i.e. the DNA is digested from the 3' or 5' end to produce a labeled nucleobase-containing monomeric degradation product.
  • the present invention is based on a cleavage reaction in the presence of a catalyst having a lower specificity, i.e. the DNA strand may be cleaved unspecifically to produce a labeled oligomeric or polymeric nucleic acid fragment.
  • the cleavage reaction is performed under spatially resolved conditions, i.e. that only the last base of the nucleic acid molecule is in contact with the catalyst.
  • the invention also relates to a unidirectional cleavage reaction comprising:
  • nucleic acid molecule to be sequenced, wherein said nucleic acid molecule is carried in an extended form by a unidirectional transport system
  • a reagent labeled with a dye e.g. a fluorescent dye wherein said fluorescent dye is capable to distinguish between nucleobases when bound thereto,
  • the unidirectional transport system comprises a flow and/or an electric field gradient.
  • the extended form of the nucleic acid molecule may be provided by terminal immobilization to a support.
  • There are multiple ways e.g. via biotin-streptavidin) to immobilize one end of the nucleic acid strand on a coated surface for subsequent manipulation in the spatially resolved cleavage reaction.
  • Possible tools for micromanipulation of the immobilized nucleic acid are: a AFM (atomic force microscopy) tip, a fiber, a microcapillary, a microcantilever or a bead, which is trapped by a electric, magnetic, optic field or by a combination field.
  • the nucleic acid molecule is immobilized at one end and the other end - to be degraded - is stretched freely in the medium, so that one can achieve a stepwise cleavage.
  • the immobilzed catalyst is contacted with the nucleic acid molecule under spatially resolved conditions and cleaves the nucleic acid molecule at a first given position. Then, the positions of the nucleic acid and/or of the catalyst have to be moved from step to step in that sense, that the degradable end of the molecule is always at the same position nearby the catalyst. In this way, spatially resolved cleavage and detection can be achieved.
  • Natural, synthetic and semisynthetic catalysts such as enzyme conjugates, imidazoles, amines and metal- or guanidinium ions [20] are known to cleave nucleic acids by hydrolysis and thus are suitable catalysts for this embodiment of the invention.
  • the labeled product can be detected and determined in a detection volume element by its nucleobase-specific signal, e.g. fluorescence characteristics.
  • the location of the detection volume element need not to be identical with the place where the cleavage occurs, but is preferably close to it in the downstream direction.
  • the dye e.g . the fluorescence dye which is used for labeling the reagent is a so-called "intelligent" dye, i.e. the dye is capable of distinguishing between different nucleobases, e.g. 2, 3 or 4 different nucleobases when bound thereto. Further, the dye is capable of distinguishing between an unbound state, e.g . the free reagent and a bound state, e.g. the degradation product.
  • the distinction between different nucleobases may be accomplished by different degrees of quenching when the dye is incorporated into a nucleoside triphosphate, e.g. when it is linked to the y-P of the released nucleoside triphosphate.
  • Suitable "intelligent" fluorescence dye molecules are known in the art.
  • the fluorescence dyes are selected from the group consisting of fluoresceines, rhodamines, oxazines, coumarines, carbostyrenes, oxadiazoles and derivatives thereof. More preferably, the fluorescence dye molecule is fluoresceine, rhodamine-6G or JF4.
  • nucleobase-specifity in fluorescence is preferably accomplished by nucleobase-specific alterations in at least one fluorescence parameter selected from fluorescence intensity, lifetime of fluorescence, anisotropy of fluorescence and/or quantum efficiency of fluorescence or any combination thereof.
  • fluorescence as used in the present application encompasses any process wherein by absorption of light an excited state in a molecule is generated, from which a light quantum is emitted, the so-called “fluorescence radiation” .
  • the nucleic acid molecule to be sequenced is e.g. a DNA molecule, e.g. a genomic DNA molecule, a cDNA molecule, a synthetic DNA molecule or any combination thereof.
  • the method of the present invention is, however, also suitable for the sequencing of RNA molecules such as mRNA molecules.
  • the nucleic acid molecule is preferably single-stranded. When sequencing double stranded molecules it should be observed that only one strand is degraded, e.g. by protecting the 3' terminus of the second strand against degradation.
  • the present invention comprises a single molecule sequencing procedure wherein the nucleotide sequence of a single nucleic acid molecule is determined.
  • a complete sequence determination may be accomplished by only one reaction, e.g. when an "intelligent" fluorescence dye is used which distinguishes between all four nucleobases.
  • fluorescence dyes are used which only distinguish between less than four bases, e.g. two or three bases or even which are specific for a single base
  • the single molecule sequencing reaction has to be performed in several parallel batches wherein the results of these parallel batches are combined, e.g. via an electronic device, in order to obtain the complete sequence.
  • a complete sequence determination is, however, not necessary, e.g. when only a partial sequence information has to be obtained, e.g. in the determination of single nucleotide polymorphisms.
  • the determination of fluorescence-labeled monomeric degradation products may be accomplished by any suitable measurement method, e.g. using a space- and/or time-resolved fluorescence spectroscopy method which is preferably capable of determining fluorescence signals which originate from a small number of molecules, e.g. from a single molecule in a small detection element.
  • the measurement may comprise confocal single molecule detection, e.g. by fluorescence correlation spectroscopy wherein a small, preferably confocal volume element is provided, having a volume of e.g. 0.1 x 1 0 "15 - 20 x 1 0 "12 I.
  • the fluorescent molecules which are located in this volume element may be subjected to the excitation light, e.g. from a laser wherein the fluorescent molecules are excited and emit fluorescent light and wherein the emitted fluorescent light originating from the volume element is measured by a photodetector.
  • EP-B-0 679 251 wherein single molecule determination by means of confocal spectroscopy is described in detail.
  • the sequencing device preferably comprises a system of microchannels, e.g. having a diameter of from 1 -1 00 ⁇ m, particularly from 1 0-50 ⁇ m.
  • the nucleic acid molecule is captured at a predetermined position in the sequencing device.
  • the capturing may comprise the use of a sealed reaction compartment, which comprises the nucleic acid to be sequenced and preferably a degradation enzyme.
  • the compartment may be sealed by a membrane which on the one hand retains the nucleic acid molecule to be sequenced, and on the other hand is permeable for released labeled products, e.g. nucleoside triphosphates.
  • the membrane is a size exclusion membrane having a cut-off value in the range of about 1 000 Da.
  • the sequencing device preferably comprises a flow reactor, wherein after the capturing of the nucleic acid the fluorescence dye labeled reagent is introduced to the sequencing device, e.g. via a continuous flow thereby starting the degradation reaction. The labeled degradation products are then passed by the flow to the detection element. Thus, the fluorescent measurement takes place downstream of the capturing position.
  • the nucleic acid molecule to be sequenced may be introduced in a carrier-bound form into the sequencing device.
  • the nucleic acid molecule is bound on a carrier particle having a diameter of preferably 0.5-1 O ⁇ m, particularly of from 1 -3 /m.
  • the carrier particle may be comprised of synthetic material such as polystyrene, glass, metals or semimetals such as silicon, metal oxides such as silica or composite materials.
  • Carrier particles containing single nucleic acid molecules may be captured by using a capturing laser, e.g. an IR laser as described in Ashkin et al. [21 ] and Chu [22]. After capturing, the labeled reagent and optionally a degradation enzyme are passed to the captured particle thereby starting the degradation. The detection may be carried out as described above.
  • a capturing laser e.g. an IR laser as described in Ashkin et al. [21 ] and Chu [22].
  • the labeled reagent and optionally a degradation enzyme are passed to the captured particle thereby starting the degradation.
  • the detection may be carried out as described above.
  • fluorescence labeled reagents particluarly fluorescence labeled phosphates or pyrophosphates are used for introducing a label into the degradation products, e.g. nucleoside diphosphates or triphosphates.
  • a further aspect of the invention relates to novel fluorescence dye labeled phosphates or pyrophosphates wherein the fluorescence molecule is coupled to the phosphate or pyrophosphate moiety via a covalent bond and preferably via a linker group.
  • the linker group preferably comprises a linear organic molecule having a chain length from at least 2 atoms, e.g. carbon atoms.
  • the linker group has a chain length of at least 3 atoms selected from carbon atoms and at least one heteroatom selected from O, P, S and/or N.
  • the fluorescence dye molecule is preferably an "intelligent" dye capable of distinguishing between different nucleobases when bound thereto, which may be selected from fluoresceines, rhodamines, oxazines, coumarines, carbostyrenes, oxadiazoles, cyanines, carbopyranines, perylenes, pyrenes, pyronines, Bodipy-dyes and derivatives thereof.
  • fluorescence labeled phosphate or pyrophosphate is of the general formula (I)
  • FI is a fluorescence dye
  • X is a bond or a linker group, contains C, Ohanded S and/or N atoms and n is an integer, preferably from 1 -30
  • Y is in each occurence independently R, S " , O " , OR or SR
  • Z is S or O and R is a monovalent ligand selected from C,-C 2 o hydrocarbon groups, e.g. aliphatic and/or cyclic alkyl, alkanyl, aralkyl or aryl groups, which may optionally contain at least one heteroatom such as halo, O, S, N, P or a salt thereof.
  • step (i) optionally coupling a linker group to a phosphate or pyrophosphate moiety, (ii) optionally purifying the product obtained in step (i), (iii) coupling a fluorescence dye to the phosphate or pyrophosphate moiety, preferably via a linker group and (iv) isolating the fluorescence dye labeled phosphate or pyrophosphate.
  • Still a further aspect of the invention is a fluorescence dye labeled nucleoside diphosphate or triphosphate, particularly a deoxyribonucleoside triphosphate wherein the fluorescence dye molecule is covalently coupled to the ⁇ -P of the nucleoside triphosphate via a linker group.
  • the linker group and the fluorescence dye are as described above.
  • the fluorescence labeled diphosphate or triphosphate is of the formula (II)
  • FI is a fluorescence dye
  • X is a bond or a linker group, contains C, O, S and/or N atoms and n is an integer, preferably from 1 -30
  • Y is in each occurence independently R, S " , O " , OR or SR
  • Z is S or O and R is a monovalent ligand selected from C C 20 hydrocarbon groups which may optionally contain at least one heteroatom such as halo, O, S, N, P and B is a nucleobase or a salt thereof.
  • the fluorescence dye labeled nucleoside diphosphate or triphosphate may be manufactured by a process comprising a phosphorolytic or pyrophosphorylytic degradation of a nucleic acid molecule in the presence of a fluorescence dye labeled phosphate or pyrophosphate as described above.
  • the degradation is preferably catalyzed by an enzyme having pyrophosphorylytic activity such as TdT.
  • the fluorescence dye labeled nucleoside diphosphate or triphosphate may be manufactured by a method comprising the steps of (i) reacting the fluorescence dye to a fluorescence dye labeled monophosphate or diphosphate, (ii) reacting the fluorescence dye labeled monophosphate obtained in step (i) with a nucleoside monophosphate or diphosphate, and (iii) isolating the fluorescence dye labeled nucleoside diphosphate or triphosphate.
  • the TdT catalyzed pyrophosphorolysis of DNA and the detection of the reaction products are described in the following sections.
  • Fluorescent dyes (Rhodamine-6G and Fluorescein) were purchased from Molecular Probes (Eugene, USA) or synthesized as described elsewhere [19]. Tris(tetra-/7-butylammonium) hydrogen pyrophosphate was purchased from Fluka. All dNDP's were purchased from Aldrich. All oligonucleotides were synthesized by Eurogentec (Belgium). [ _32 P] ATP was purchased from New England Biolabs. All other chemicals were purchased from Fluka or Aldrich. Calf thymus TdT and T4 polynucleotide kinase were purchased from Roche. QAE-Sephadex A25 was purchased from Pharmacia. Dowex 50W-X8 (H + ) was purchased from Fluka. Ultrafiltration membranes and Microcon centrifugal filter devices were purchased from Millipore.
  • 4.6 g (1 9.1 mmol) of 2 was dissolved in 60 ml dry CH 2 CI 2 , 4 ml (28.7 mmol) of triethylamine was added, and the solution was cooled to 0 ° C.
  • 1 .8 ml (23 mmol) of methanesulfonyl chloride was slowly added to the cooled solution of 2. The solution was stirred for 1 h at 0 ° C and washed with saturated aqueous NaHCO 3 .
  • the product was dissolved in 7.5 ml of 20 mM TEABC buffer, pH 9, and loaded onto the column.
  • a gradient of 1 M TEABC buffer, pH 9 was run (buffer A: 20 mM TEABC, buffer B: 1 mM TEABC, 0 to 40% B over 200 ml of mobile phase, 40 to 70% B over 1000 ml, and 70 to 1 00% B over 1 1 00 mi, flow rate: 2.5 ml/min), and the fractions were detected by UV at 260 nm
  • the UV active fractions were checked by 31 P NMR. Those containing pure product were pooled and evaporated to give 2.1 g (62% yield) of triethylammonium salt of 4.
  • the JF4 pyrophosphate was purified by reverse phase HPLC (RP-18, 8 x 250 mm; eluant: H 2 O (A), acetonitrile (B), a gradient of 0 to 60% B in 60 min, flow rate: 3 ml/min, t R 33.5 min). The collected fractions were lyophilized to yield 1 mg (13%) of 6b.
  • the reactions were performed under argon.
  • the fluorescent dye labeled monophosphates were transferred in a tri-/7-butylammonium salt as described by Hoard and Ott [26].
  • the fluorescent dye . labeled monophosphate tri-n-butylammonium salt (0.35 //mol, 1 eq) was dissolved in 0.5 ml DMF and placed in a flame-dried flask sealed with a silicone septum.
  • the solution of 1 .75 //mol (5eq) 1 , 1 '-carbonyldiimidazole in 0.5 ml DMF was slowly added and the reaction mixture was stirred at room temperature for 1 day.
  • 5 '- 32 P-Iabeled oligonucleotides were prepared using T4 polynucleotide kinase and [ ⁇ 32 P]ATP as the phosphate source.
  • the reaction mixture contained, in a final volume of 50 //I, 50 mM Tris buffer, pH 8.2, 1 0 mM
  • the radioactive labeled oligonucleotide was separated from unincorporated [ - 32 P]ATP by using a Sephadex G-25 column and sterile water as eluent.
  • the oligonucleotide (24mer) was purified from shorter and longer nucleotides by urea-PAGE and crush and soak to recover the oligonucleotide. Bands were located by UV-shadowing, excised and extracted into 0.1 % SDS, 0.5 M ammonium acetate and 1 0 mM MgCI 2 overnight.
  • the oligoirucleotide was desalted on a Microcon YM-3 filter using water for washing, followed by a sterile Sephadex G-25 column with water as eluent.
  • Radioactive fractions were collected in 1 .5 ml microcentrifuge tubes and monitored using a Geiger counter. Only the radioactive fractions were kept for further experiments. The final concentration of the radioactive labeled oligonucleotide was 50-100 / M.
  • Each reaction mixture contained, in a final volume of 20 ⁇ at pH 7.8, 50 mM Hepes buffer, 2 mM MgCI 2 , 20 mM KCI, 5 ⁇ U 5 '-[ 32 P] oligonucleotide and 50 U TdT. An aliquot of the respective pyrophosphate solution was added to yield the final concentration of 1 mM. The reaction was carried out at 37 ° C. After 0, 30, 60 and 120 min, 5 ⁇ were taken and added to 5 ⁇ of stop solution (95% formamide/0.05% bromophenol blue) .
  • reaction mixture was kept at 100 ° C for 3 min to denature the TdT and then terminate the reaction.
  • the reaction volume was then concentrated on a sterile Microcon YM-3 filter to 10 ⁇ . 1 ⁇ was loaded onto a 1 9% polyacrylamide gel containing 8M urea and submitted to electrophores is at 2500 V.
  • the reaction with Fluorescein pyrophosphate 6a was performed under the same conditions in the reactor; the only difference being that the pyrophosphorolysis buffer contained only 0.2 mM Fluorescein pyrophosphate and the reaction was stopped after 2 hours.
  • the fluorescence properties (see definitions below) of fluorescent dyes can be influenced by the environment either by unspecific effects determined by the polarity of the surrounding medium or by specific effects caused by molecular interactions with neighboring molecules or groups.
  • fluorescent dyes such as xanthene or coumarine dyes
  • a dye that shows a significant different fluorescence behavior with different nucleobases is called an "intelligent" dye.
  • a differently strong quenching effect from a nucleobase to a fluorophore enables the identification of the nucleobase.
  • Coumarin 1 20 is such a fluorophore, but is less preferred for single molecule detection because of its low photostability.
  • Rhodamines are more preferred for single-molecule detection, but the fluorescence of the fluorophores rhodamine 6G (Rh6G) and tetramethylrhodamine (TMR) is only quenched by guanine and not by the other three nucleobases.
  • Single molecule spectroscopy enables us to detect simultaneously several fluorescence parameters, i.e. fluorescence intensity, fluorescence lifetime and anisotropy. This can be realized with the method of multiparameter fluorescence detection (MFD), explained in detail below.
  • MFD allows one to resolve inhomogeneities and sub-states in a sample containing molecules with fluorescent properties.
  • the sequencing method on single molecule level allows the sorting out of each labeled nucleotide and to carry out a stepwise MFD analysis.
  • Fluorescence is the deactivation process from the lowest vibrational level of an electronically excited state of a fluorophore to the ground state after excitation with light and under emission of a photon . This process can be described by different parameters: quantum yield, intensity, lifetime, anisotropy and spectral (energetic) properties of excitation and emission.
  • the fluorescence quantum yield ⁇ F is the ratio of the number of emitted photons to the number of absorbed photons.
  • _ F number o 1f emitted i p. —hot .ons ⁇ ⁇ ,1 number of emitted photons Eq . 1 a
  • the estimation of fluorescence quantum yield of a sample dye relative to a reference molecule with a known fluorescence quantum yield follows the Eq . 1 b
  • F stands for the fluorescence intensity respectively the area under the fluorescence spectrum and optical density, OD, measured by an absorption spectrometer.
  • N(t) the number of excited molecules at time t after excitation
  • Eq. 2 relates it to the number of excited molecules at time 0, N 0 , and the rate constant of radiative deactivation k F :
  • the fluorescence lifetime r F is defined as time t, during that the number of excited molecules has dropped to ⁇ 0 /e.
  • the lifetime is inverse proportional to the fluorescence rate constant and is the time of the molecule being in the excited state until it fluoresces and returns to the ground state.
  • r F is called the "radiation lifetime" because for the experimentally obtained lifetime additional processes have to be taken into account such as internal conversion, IC, a radiationless relaxation process, and intersystem crossing, ISC, a process between triplet and singlet states of a molecule.
  • the parameter anisotropy r gives an estimate for the local mobility of the dye in the sample. The values vary between -0.2 and 0.4. The higher r, the less mobile the dye. The anisotropy is related to the fluorescence lifetime over the Perrin equation
  • the electronic energy of the excited state can be transferred from the fluorophore to a quencher.
  • DQ non-fluorescent complexes
  • the complex DQ can emit photons with another wavelength compared to the free fluorophore or can return into the ground state without emission of light.
  • Dynamic quenching is caused by collisions between the excited fluorophore and the quencher.
  • dynamic quenching is also called collisional quenching.
  • the decrease in fluorescence lifetime follows the equation
  • the fluorescence quantum yield and therefore the fluorescence lifetime are proportional to the intensity F of a fluorescence band.
  • the ratio of fluorescence lifetime and intensity in the presence (r and F) and absence (r 0 and F 0 ) of quencher follows Eq. 8.
  • the pinhole had a diameter of 1 00 //m.
  • the dichroic mirror was 510DCLP and fluorescence filters HQ575/70 and HQ730/140 were used.
  • the samples were measured in phosphate buffer (1 0 mM Na 2 HP0 4 /NaH 2 PO 4 , 1 80 mM NaCl, pH 7.46) at a temperature between 20 and 25 ° C.
  • the concentration of each sample was between 50 and 100 pM.
  • Individual molecules passing through the open volume element are readily detected by their brief fluorescence bursts and selected from the background signal.
  • the fluorescence signal is divided into its parallel and perpendicular components with respect to the linear polarized excitation beam by a polarizing beam splitter cube, which is then subsequently divided into "green" (wavelength ⁇ ⁇ 595 nm) and "red” ( ⁇ > 595 nm) fluorescence components by dichroic mirrors resulting in four signal paths. Each burst is analyzed to determine a single set of dye-specific fluorescence parameters.
  • the synthesis of the fluorescent labeled pyrophosphates is summarized in Figure 2.
  • the route is flexible with respect to the usage of several fluorescent dyes, employing simple well established coupling techniques based on the formation of amide bonds and allowing for adjusting the nature of the linker.
  • An aminoethoxyethyl linker 1 was used to connect the fluorescent dyes with the pyrophosphate group. This mobile, water-soluble linker warrants better acceptance of the dye labeled pyrophosphate by the enzyme for sterical reasons.
  • good stacking of the base with the fluorophore at the stage of the labeled triphosphate is necessary to allow differentiation of the four nucleobases based on different interaction with the fluorophore.
  • the functional groups of 1 , amino group and hydroxyl group can easily be substituted by activated fluorescent dyes and pyrophosphates. This is a versatile approach to synthesise different dye labeled pyrophosphates because one can easily vary the length of the linker and the dye.
  • the pyrophosphate ester 5 is the key reagent in the synthesis of fluorescent labeled pyrophosphates, and is prepared from the aminoethoxyethanol 1 in five steps.
  • the amino group has been protected using the benzyloxycarbonyl protecting group. This prevents possible cyclisation, when the carbonyl group is activated by mesylate during the next step.
  • the benzyloxycarbonyl group can be detected by UV light which simplifies the purification of the pyrophosphate.
  • the carbonyl group of 2 was activated with methanesulfonyl chloride and 3 was pyrophosphorylated using twofold excess of tris(tetra- ⁇ -butylammonium) hydrogen pyrophosphate [23, 24] .
  • 3 was pyrophosphorylated using twofold excess of tris(tetra- ⁇ -butylammonium) hydrogen pyrophosphate [23, 24] .
  • a complete removal of inorganic pyrophosphate from the pyrophosphate ester 4 is performed, since the inorganic pyrophosphate would later disturb the enzymatic reaction with the dye labeled pyrophosphate.
  • Purification of the pyrophosphate ester 4 is advantageous because of the UV activity of the amino protecting group. 31 P-NMR of 4 proves that the inorganic pyrophosphate was completely removed (Figure 3).
  • pyrophosphate 5 For the labeling of pyrophosphate 5 with the fluorescent dye Fluorescein we used its succinimidyl ester. For the dyes Rhodamine-6G and JF4, their carboxyl group was activated by the method described by Gillessen and co-workers [25] . This reaction requires a water free solvent that prevents hydrolysis of the intermediates. DMF was most suited due to the high solubility of fluorescent dyes in this polar, aprotic solvent. The pyrophosphate 5, however, is not soluble in DMF, whereas its tri-/7-butylammonium salt that is obtained by exchange of triethylammonium by tri-/7-butylammonium [26] . The resulting tri-/7-butylammonium salt of pyrophosphate 5 was reacted with the activated fluorescent dyes to give fluorescent dye labeled pyrophosphates 6a-c.
  • the fluorescent dye labeled pyrophosphates were used in the pyrophosphorolysis reaction of DNA.
  • the purity of the fluorescent dye labeled pyrophosphates was ensured by HPLC and 31 P NMR to exclude the contamination with inorganic pyrophosphate and/or with dNTP ' s.
  • the evaluation of the pyrophosphorolysis reaction can in principle be done by detection of the dye labeled dNTP ' s or the modified DNA.
  • the radioactively labeled DNA was purified before use in the pyrophosphorolysis reaction by denaturing gel electrophoresis.
  • the DNA was visualised by UV absorption, excised and retrieved from the gel using "crush and soak” techniques as described in materials and methods.
  • the purified DNA was desalted twice on Sephadex G-25 columns to completely remove the SDS contained in the elution buffer.
  • the purified, 32 P-5 '- labeled DNA was incubated for 1 h with TdT and the various pyrophosphate derivatives in the presence of 1 x TdT buffer containing 2 mM Mg 2+ , 20 mM KCI and 50 mM HEPES buffer (pH 7.8) .
  • the concentration of inorganic pyrophosphate, Fluorescein-pyrophosphate 6a and Rhodamine-6G-pyrophosphate 6b was 1 mM.
  • the concentration of JF4-pyrophosphate 6c was 0.2 mM due to its low solubility.
  • the reaction products were analyzed in a 61 cm gel electrophoresis apparatus with 1 9% polyacrylamide gel containing 8 M urea.
  • the elongated oligonucleotides require the presence of dNTP ' s which can only have been formed by pyrophosphorolysis and reincorporation at the 3 '-end of oligonucleotides.
  • the equilibrium between the degradation versus incorporation reaction depends on the nature of the dye. While for JF4 the ratio is approximately 1 : 1 , despite the smaller pyrophosphate concentration, the polymerisation products dominate for Fluorescein pyrophosphate 6a and even more for the Rhodamine-6G substituted pyrophosphate 6c. With inorganic pyrophosphate both the pyrophosphorolysis activity of TdT and the overall rates are maximal.
  • the flow reactor consists of a cylindrical reaction chamber and two covers with holes for the supply and drain of the pyrophosphate containing 1 x TdT buffer (see Figure 5). On the top and the bottom of the reaction chamber two membranes with cut-off volumes of 1 000 Da are placed that are permeable only for low molecular weight compounds like pyrophosphates and dNTP 's but not for DNA and TdT.
  • reaction chamber was fasten with screws, after addition TdT, DNA and 1 x TdT buffer without inorganic pyrophosphate.
  • a solution of inorganic pyrophosphate in 1 x TdT buffer is then pumped through the reactor using a HPLC-pump at flow-rates up to 40 //l/min.
  • the pyrophosphorolysis reaction with the flow reactor was carried out using inorganic pyrophosphate, Fluorescein-pyrophosphate 6a and no pyrophosphate as control.
  • the same reactions were performed in regular 1 .5 ml reaction caps.
  • the reactions were analyzed using urea-PAGE as described previously with radioactively detection. The results are presented in Figure 6.
  • For the control reactions no length change of oligonucleotides is observed, neither in the cap ( Figure 6, lane 4) nor in the flow reactor ( Figure 6, lane 3).
  • phosphate ester 7 ( Figure 7). Unreacted phosphoric acid was precipitated as lithium salt and separated by filtration. The lithium salt of phosphate ester 7 was transformed to the tri-/7-butylammonium salt and reacted with the activated fluorescent dye in the same way as for the pyrophosphates ( Figure 2).
  • the purification and the handling of the dye labeled phosphates 8 and 9 is significantly easier than the purification of the dye labeled pyrophosphates, since they cannot hydrolyse.
  • the fluorescent dye labeled phosphates 8 and 9 were then converted to the y-P-fluorescent-dye-labeled dNTP ' s as shown in Figure 7 using the phosphoester-forming reaction as described by Cramer [27] .
  • the fluorescent dye labeled phosphates 8 and 9 were activated using the
  • Table 1 summarizes the Stern-Volmer quenching constants from different fluorophores (i.e. rhodamine and oxazine dyes) with the four nucleotides dAMP, dCMP, dGMP and TMP. The measurements are taken in acetate buffer (50 mM sodium acetate, pH 5.5) .
  • dAMP quenches the fluorescence from cyanorhodamine, from the listed oxazines and from rhodamine 1 1 0 and 1 23.
  • the quenching effect by dAMP is smaller than by dGMP, which corresponds to the more positive oxidation potential of adenosine.
  • the fluorescence parameters shown here in the example are: fluorescence intensity in the green detection channels, S G , the intensity ratio from the green and the red detection channels, S G /S R , fluorescence lifetime, T, and anisotropy, r.
  • the frequency of parameter pairs equal to the number (#) of fluorescence bursts, are counted in 2D histograms with the corresponding 1 D histograms given as projections (figures 8 to 1 0) .
  • the sample Rh6G-dGTP contains two species: According to the results shown in figure 8, one belongs to the pure dye.
  • the other compound is the dye with linked nucleoside-triphosphate.
  • the fluorescence lifetime of the dye is shortened with a concomitant decrease of the fluorescence intensity.
  • the third parameter, the anisotropy shows a better resolution of the two species. Therefore, the differentiation of the two species is possible, avoiding further purification of the sample.
  • Rh6G-dATP seems to be mostly homogeneous and can be distinguished from the sample with Rh6G-dGTP.
  • the fluorescence lifetime of the species is increased compared to the lifetime of the pure dye.
  • Figure 1 The concept of DNA sequencing by TdT catalyzed pyrophosphorolysis.
  • a single DNA strand is incubated with TdT and pyrophosphate labeled with "intelligent" dye.
  • the DNA is degraded sequentially by the pyrophosphorolysis reaction and deoxynucleotide 5 '-triphosphates labeled at y-P with the intelligent dye are formed during the degradation.
  • the formed ⁇ -P fluorescent dye labeled dNTP ' s can be detected and identified by confocal fluorescence microscopy by virtue of their different fluorescence properties.
  • Figure 2 Synthesis of the fluorescent dye labeled pyrophosphates 6a-c. i: N-(Phenylmethoxy-carbonyl)-2-(2-aminoethoxy)ethanol, dioxan, 0 ° C, RT, 24 h; ii: methanesulfonyl chloride, Et 3 N, CH 2 CI 2 , 0 ° C, 30 min; iii: (Bu 4 N) 3 HP 2 0 7 , acetonitrile, RT, 1 8 h; iv: QAE-Sephadex A-25, 1 M TEABC buffer (pH 9), 20 mM TEABC buffer (pH 9); v: H 2 /Pd(C), H 2 0, RT, 3 h; vi: R-COOH, TBTU/DIEA or R - succinimidyl ester, DMF; HPLC purification.
  • i N-(Phenylmethoxy-carbony
  • Figure 3 Purification of pyrophosphate 4 as determinate by NMR spectroscopy (400 MHz), 300 K in D 2 0 (pH 8).
  • (a) Decoupled 31 P spectrum of 4 before the chromatographic purification; the singlet at ⁇ -6.5 ppm correspond to inorganic pyrophosphate;
  • FIG. 4 Pyrophosphorolysis of 24mer DNA with inorganic pyrophosphate and fluorescent dye labeled pyrophosphates.
  • TdT was incubated with the 24mer at 37 ° C and pH 7.8 in a 20 ⁇ volume containing 50 mM Hepes buffer, 2 mM MgCI 2 , 20 mM KCI, 5 ⁇ 5 ' -[ 32 P]-labeled oligonucleotide and different pyrophosphates.
  • Lane 1 shows the oligonucleotide products after the pyrophosphorolysis with 1 mM inorganic PPi; lane 2: with 1 mM Fluorescein-PPi (6a); lane 3 with 0.2 mM JF4-PP (6b); lane 4: with 1 mM Rhodamine-6G-PPi (6c); lane 5 is a control lane in the absence of pyrophosphate.
  • B slices trough the corresponding lanes.
  • FIG. 5 Detailed view of the flow reactor.
  • the reactor consists of a cylindrical reaction chamber (500 //I) made up of two steel blocks (1 and 2) with openings on the top and bottom for supply and release.
  • the reactor chamber was sealed by O-seals and fasten with screws.
  • the supply and release tubes were connected to the reactor using screw-in joints.
  • the TdT and DNA are confined in the reaction chamber by two membranes with cut-off volumes of 1000 Da.
  • the membranes are placed on the support between the opening and the chamber on both sides of supply and release.
  • the cylindrical chamber and membrane supports were coated with Teflon to avoid contact of TdT and DNA with metal.
  • the flow was controlled by HPLC pump.
  • Figure 6 Demonstration of the pyrophosphorolysis in the flow reactor (lanes 3 and 6) in comparison with the pyrophosphorolysis reaction carried out without flow in 1 .5 ml cap (lanes 1 , 4, 5).
  • Lane 3 and 4 are the results from the control reactions carried out in the absence of pyrophosphate in the flow reactor and in the 1 .5 ml cap, respectively.
  • Lane 1 represents the results in the presence of 1 mM PPi after 5h, and lane 5 in the presence of 0.2 mM Fluorescein-PPi (6a) after 2 h, both in 1 .5 ml caps.
  • Lane 2 stems from the reaction with 1 mM PPi after 5h and lane 6 with 0.2 mM Fluorescein-PPi after 2 hours both in flow reactor. From the two lanes stemming from the reaction in the flow reactor it is obvious that a full degradation of DNA with dye labeled pyrophosphate can be achieved.
  • Figure 7 Chemical synthesis of fluorescent dye labeled dNTP ' s using the 1 , 1 '-carbonyldiimidazole method, i: The reaction was kept in vacuum at 1 50 ° C for 1 8 h; ii: 5 M LiOH solution was added up to pH 1 0.5; iii: Dowex 50W-X8 (pyridinium); tri-n-butylamine, DMF, 67%; iv: Rhodamine-6G succinimidyl ester, DMF, RT, 4 days; HPLC purification; Dowex 50W-X8 (pyridinium form), Bu 3 N, DMF; v: 5-carboxy-JF4, TBTU/DIEA, RT, 4 days; HPLC purification; Dowex 50W-X8 (pyridinium form), Bu 3 N, DMF; vi: 1 , 1 '- carbonyldiimidazole, DMF, RT, 1 day; vii: MeOH, RT, 45 min
  • Figure 8 Two-dimensional histograms of the fluorescence parameters of the fluorophore rhodamine 6G (Rh6G) at the single molecule level.
  • A Fluorescence lifetime ⁇ vs. intensity ratio F D /F A .
  • B Fluorescence lifetime r vs. anisotropy r G .
  • C Fluorescence liftetime t vs. fluorescence intensity S G .
  • Figure 9 Two-dimensional histograms of the fluorescence parameters of Rh6G-dGTP at the single molecule level.
  • A Fluorescence lifetime r vs. intensity ratio F D /F A .
  • B Fluorescence lifetime r vs. anisotropy r G .
  • C Fluorescence lifetime r vs. fluorescence intensity S G .
  • Figure 10 Two-dimensional histograms of the fluorescence parameters of Rh6G-dATP at the single molecule level.

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Abstract

L'invention concerne un procédé de séquençage d'acides nucléiques selon lequel la molécule d'acide nucléique à séquencer est dégradée de manière séquentielle en présence d'un réactif marqué par fluorescence, un nucléoside marqué par fluorescence étant formé et présentant des caractéristiques de fluorescence spécifiques d'une nucléobase. De préférence, ce procédé est mis en oeuvre par une procédure de séquençage de molécule unique qui comprend une étape de détection résolue de manière spatiale, par exemple de détection confocale. De préférence, ce procédé comprend une étape de détection confocale. L'invention concerne en outre des nouveaux colorants fluorescents spécifiques d'une nucléobase et des réactifs contenant ces colorants.
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JP2003189852A (ja) * 2001-12-26 2003-07-08 Olympus Optical Co Ltd 塩基配列決定装置および塩基配列決定方法
WO2014167324A1 (fr) * 2013-04-09 2014-10-16 Base4 Innovation Ltd Méthode de détection de mononucléotide
WO2014167323A1 (fr) * 2013-04-09 2014-10-16 Base4 Innovation Ltd Procédé de détection de nucléotides simples
WO2014187924A1 (fr) * 2013-05-24 2014-11-27 Illumina Cambridge Limited Sequençage pyrophosphorolytique
JP2015521030A (ja) * 2012-04-09 2015-07-27 ザ・トラスティーズ・オブ・コランビア・ユニバーシティー・イン・ザ・シティー・オブ・ニューヨークＴｈｅ Ｔｒｕｓｔｅｅｓ Ｏｆ Ｃｏｌｕｍｂｉａ Ｕｎｉｖｅｒｓｉｔｙ Ｉｎ Ｔｈｅ Ｃｉｔｙ Ｏｆ Ｎｅｗ Ｙｏｒｋ ナノ細孔の調製方法およびその使用
JP2015530119A (ja) * 2012-10-04 2015-10-15 ベース４ イノベーション リミテッド 配列決定方法
CN105518152A (zh) * 2013-06-13 2016-04-20 贝斯4创新公司 液滴储存方法
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