WO1999013110A1 - Procede de fabrication de photoproduits de nucleotides fluorescents pour sequençage d'adn et analyse - Google Patents

Procede de fabrication de photoproduits de nucleotides fluorescents pour sequençage d'adn et analyse Download PDF

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WO1999013110A1
WO1999013110A1 PCT/US1998/018817 US9818817W WO9913110A1 WO 1999013110 A1 WO1999013110 A1 WO 1999013110A1 US 9818817 W US9818817 W US 9818817W WO 9913110 A1 WO9913110 A1 WO 9913110A1
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nucleotides
fluorescence
nucleotide
solution
products
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PCT/US1998/018817
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John J. Macklin
Jay K. Trautman
Timothy D. Harris
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Seq, Ltd.
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Priority to AU94765/98A priority Critical patent/AU9476598A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61NELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00Radiation therapy
    • A61N5/06Radiation therapy using light
    • A61N2005/065Light sources therefor
    • A61N2005/0651Diodes
    • A61N2005/0653Organic light emitting diodes

Definitions

  • the present invention relates to the formation of fluorescent products of native nucleotides as a result of a photochemical reaction, and the use of these photoproducts for enhanced detection of nucleotides by fluorescence. It additionally relates to the binding of these nucleotides to surfaces, where immobilization facilitates their detection at exceedingly low concentrations.
  • One such method to sequence DNA takes advantage of the sequential cleavage of bases from an oligonucleotide by a processive exonuclease, where the sequence is determined by the detection and discrimination of the ordered cleaved bases.
  • Such a base-at-a-time sequencing device operated at high throughput (10-100 bases/sec) on long DNA strands (20-40 kilobase lengths) , depends fundamentally on the reliable fluorescence detection of nucleotides at the single molecule level.
  • nucleotide fluorescence quantum yield the fractional number of fluorescent photons emitted per absorbed photon
  • bleaching is probabilistic, in order to insure that, say, 90% of the molecules survive the bleaching process to yield at least 4000 photons, the actual bleaching probability should be less than about 2 x 10 ⁇ 5 . Any analysis of single molecule detection which fails to address this photostability issue will arrive at fundamentally flawed conclusions.
  • the four common nucleotides are virtually non-fluorescent under physiological conditions, such as those where a single DNA strand is to be sequentially cleaved by an exonuclease, with quantum yields in the range of 0.01%. Only at acid pH will G and to a much smaller degree A show any fluorescence, with a typical quantum yield for G of 2% in a room temperature, aqueous solution at pH 1.68. This should be referred to as an average quantum yield, since the fluorescence decay is not a single-exponential, indicating a distribution of quantum yields. The quantum yields of the four nucleotides preclude single molecule detection in room temperature solutions.
  • nucleotide fluorescence can be substantially increased, with quantum yields of about 15% for G and T, 5% for C, and ⁇ 1% for A.
  • G in a room temperature, low pH glass formed by a spin-coating method has an apparent quantum yield of about 15%, which increases to about 50% at 77 °K.
  • Other nucleotides also show increased yield, for example T is 1% in a room temperature glass, which increases to about 8% at 77 ° . So the enhancement of quantum yield of nucleotides is mainly due to the rigidity of the matrix, but an additional improvement is obtained at very cold temperatures.
  • the molecule spends in the metastable triplet state it can react with the surrounding matrix, and decompose as a result, or it can absorb some of the incident uv light, and get promoted to a new state which can ionize or dissociate, again leading to decompostion and irreversible bleaching.
  • the rate of these decomposition processes in the former case is linear in uv intensity while in the latter case it depends on the square of the intensity.
  • the nonlinear bleaching can be mitigated by lowering the intensity, and we have demonstrated a method to perform simultaneous detection of many individual molecules in an imaging detection arrangement, which allows substantial reduction in excitation intensity without loss in sequencing speed (i.e., the number of detected nucleotides per second). For the bleaching that is linear in intensity, no change in uv intensity will mitigate the bleaching, because the fluorescence is linear in intensity also.
  • triplet state can be quenched by other molecules through an energy transfer process, and this reduces the triplet lifetime and therefore the chance a decomposition occurs.
  • oxygen is a triplet quencher, but is potentially undesirable because when oxygen accepts energy from a nucleotide triplet state, it may form a radical which subsequently attacks and decomposes the nucleotide.
  • Other quenchers are known, for example, hexadien-1-ol, which appear to not form radicals.
  • a key physical condition for effective triplet quenching is the proximity and orientation of the quencher molecule with respect to the triplet-excited nucleotide.
  • the emission rate is limited to approximately QY/ (t 2 + P isc t tr ⁇ plet ) , where P 1SC is the probability to populate the triplet and typically 0.1 for the nucleotides, t triplet is the lifetime of the triplet state and typically 0.3 sec at 77 °K, t 2 is the emitting state lifetime, typically 10 ⁇ 8 sec, and QY is the fluorescence quantum yield of the nucleotide, say 15%.
  • the emission rate is then about 5 photons/sec. With a collection efficiency of 2% (typical) , it would take 1000 sec to record a total of 100 photons per nucleotide. There would be little competitive advantage to single-molecule sequencing under this set of circumstances.
  • nucleotides to enhance the fluorescence properties, principally increased quantum yield, and resistance to bleaching or decomposition, i.e., increased photostability.
  • An additional requirement for a sequencer is that the modified nucleotides can be discriminated, by either a spectroscopic characteristic such as absorption or emission maxima, fluorescence lifetime, or a physical property which in the presence of a driving force, leads to a characteristic response, such as electrophoretic mobility.
  • Modification of the nucleotides prior to the sequential enzymatic cleavage step is undesirable as it is time-consuming, has not been shown to work with a processive enzyme, and can introduce errors in the sequence under study.
  • An alternative involves modification of the nucleotide following the cleavage step, either while the nucleotides are freely diffusing toward a binding surface, or after they are immobilized on a binding surface.
  • a solution containing a native nucleotide and a non- fluorescent reagent is illuminated with electromagnetic radiation which results in a photochemical reaction that yields a fluorescent analog of the native nucleotide. This fluorescent photoproduct is then useful for the quantitative analysis of the native nucleotide by fluorescence detection methods.
  • a photochemical reaction is considered to consist of the following microscopic processes: (1) absorption of light resulting in the formation of an electronically excited state, either of the nucleotide or of the reagent, (2) primary photochemical reaction involving the excited nucleotide and reagent, or excited reagent and nucleotide, that form intermediate molecules, followed by (3) thermally- driven reactions of the intermediates to stable products.
  • this method can be a highly specific means to essentially fluorescently-label native nucleotides.
  • this reaction can be run on nucleotides bound to and immobilized on a surface, or while nucleotides diffuse to such a surface.
  • the nucleic-acid components preferably are the 5'- onophosphate nucleotides (dAMP, dCMP, dGMP and TMP) .
  • dAMP 5'- onophosphate nucleotides
  • dCMP 5'- onophosphate nucleotide
  • dGMP 5'- onophosphate nucleotide
  • TMP 5'- onophosphate nucleotide nucleotides
  • these nucleotides are sequentially cleaved one base at a time from an oligonucleotide by a processive enzyme, and immobilized on a surface in the order in which they were cleaved, so that spatially-resolved fluorescence detection and identification of single nucleotides can then be used to determine the original sequence of cleaved nucleotides, and thus the DNA sequence of the original oligonucleotide.
  • the reagents to be used in the photochemistry are organic molecules such as alcohols, amines, or other hydrogen-atom donors, that are ideally non-fluorescent, so as to not obscure or limit the detection of fluorescence from the nucleotide photoproduct. Since photochemistry from reactants to products often takes place via radical intermediates, formed initially by hydrogen- or electron- abstraction from a donor reagent molecule, reagents for photochemistry may be chosen from a list of hydrogen-atom donating molecules that includes alcohol-, sugar-, amine-, hydrocarbon-containing compounds, and this list includes nucleic acid components as well.
  • reagents that are good hydrogen-atom acceptors such as aromatic ketones like benzophenone, may also be useful.
  • the principal issue in the choice of the reagent is the specificity of the photochemical reaction to produce a fluorescent product of a nucleotide, while additionally that product should not undergo photochemical reactions itself.
  • photochemical reactions usually proceed from the triplet state of a molecule.
  • a long-lived triplet state is desirable to increase the time for encounters with reagent molecules.
  • oxygen is a known triplet quencher
  • nitrogen-purged or otherwise anaerobic solutions as well as methods to enhance the intersystem crossing rate, should increase the photochemical quantum yield.
  • the conversion of a native nucleotide to a fluorescent product is accomplished using electromagnetic radiation to selectively deposit energy in the reactant molecules, and initiate the subsequent dark (or thermal) chemical reactions.
  • Photochemical reactions and more generally reactions proceeding from a molecular excited state, can access states which are energetically unfavorable in thermal reactions at any realistic temperature, while minimizing undesirable side reactions.
  • the detection of single nucleotides that have been photochemically modified can be facilitated by immobilization on a surface.
  • a convenient and general binding motif for the nucleotides is the electrostatic attraction between the monophosphate and the surface ions of a metal-oxide film. This binding arrangement can immobilize nucleotides for hours to days, and can be shown to have little deleterious effect on the fluorescence properties of the bound nucleotides.
  • the surface-bound molecules can be advantageously excited using a total-internal-reflectance geometry, which minimizes excitation and hence background fluorescence of the solvent.
  • the nucleotide fluorescence can be detected in either a full- field imaging, or by using confocal detection.
  • FIG. 1 is a flowchart depicting a preferred embodiment of the invention
  • FIGs. 2(a) and 2(b) depict experimental setups used in the practice of the invention
  • Figs. 3(a) through 3(f) are plots of absorbance and fluorescence intensity versus wavelength
  • Figs. 4(a) and 4(b) are plots of absorbance and fluorescence intensity versus wavelength;
  • Figs. 5(a) and 5(b) are plots of absorbance and fluorescence intensity versus wavelength;
  • Figs. 6(a) and 6(b) are plots of fluorescence intensity versus wavelength;
  • Fig. 7 is a plot of photocounts versus time;
  • Fig. 8 is a plot of fluorescence intensity versus pH
  • Fig. 9 is diagrams of several molecules
  • Figs. 10(a) and 10(b) are plots of fluorescence intensity versus time;
  • Fig. 11 is a plot of photocounts versus time;
  • Figs. 12(a) and 12(b) are CCD images and linecuts of single nucleotides on a surface.
  • Fig. 1 is a flowchart depicting a preferred embodiment of the invention.
  • a nucleotide is first contacted with an essentially non-fluorescent reagent.
  • the nucleotide and reagent are then illuminated with electromagnetic radiation, typically in the ultraviolet part of the spectrum, to form a conversion product.
  • the conversion product is then illuminated with electromagnetic radiation to stimulate fluorescence.
  • the fluorescence is detected by a suitable detection system, typically a CCD camera, a spectrometer-coupled CCD or a microchannel plate detector.
  • the conversion product should have a quantum yield of at least 10% and it should emit at least 1000 photons before photo bleaching.
  • the quantum yield and the photon emission should be much higher with the quantum yield about 50% and the number of photons emitted before photo bleaching in the range of 10,000.
  • Figs. 2(a) and 2(b) Photochemistry of nucleotides, and analysis of the fluorescent products, was performed using the experimental arrangements shown in Figs. 2(a) and 2(b).
  • large quantities of photoproduct are made by uv-illuminating a cuvet containing native nucleotides in an aqueous alcohol solution.
  • the apparatus comprises an ultraviolet laser 70, a lens system 72, a quartz cuvet 74, a gas inlet 76 and a vent 78.
  • This arrangement allows us to isolate the conditions that optimize the photochemistry process from those that optimize the fluorescence properties of the nucleotide photoproduct (the quantum yield, photostability, spectral characteristics, etc) , since the cuvet containing the solution can be conveniently removed and analyzed for changes in absorbance and fluorescence using a spectrophotomer and a luminescence spectrometer (not shown) .
  • the change in absorbance versus the absorbed uv energy then gives the photochemical quantum yield (the probability of converting a nucleotide molecule into a photoproduct molecule per absorbed uv photon) .
  • These bulk solutions were used as stock solutions from which aliquots of photoproduct could be taken and tested versus pH, etc.
  • This apparatus comprises one or more sources of a beam of ultraviolet radiation (not shown), a lens system 102, a quartz substrate 104 bearing a thin film solution 106, a CCD camera 108, a spectrometer 110 coupled to a second CCD camera 112, a uv-enhanced microchannel plate detector 114, a beam- splitter 116, removable mirrors 118, 120, and filters 122, 124.
  • This arrangement was also used to measure the fluorescent properties of nucleotides bound to a surface at a liquid/solid interface. Either epi-illumination or evanescent-wave excitation was used. For epi-illumination all of the solution can be excited, while for evanescent- excitation, only molecules on or within about 0.03-microns of the quartz surface are excited. Photoproduct fluorescence was monitored by either the CCD camera, onto which the sample fluorescence was imaged, by the spectrometer -coupled CCD for spectrally-resolved fluorescence measurements, and by the microchannel plate detector, for time-resolved measurements. Nucleotide monophosphates were purchased from Sigma or Aldrich (>99% purity) and used as received.
  • the principal source of ultraviolet light was a Ti: sapphire laser, frequency-tripled into the uv, that produced tunable wavelengths from 260 nm up to 295 nm with 100-200 mWatts of power.
  • Other sources used included an Ar- ion laser that produced 3 mW at 275 nm and 10 mW at 300 nm.
  • Fluorescence lifetime of samples after photoconversion was determined by time correlated photon counting, using the frequency-tripled, mode-locked Ti: sapphire laser with a pulse repetition rate of 82 MHz and a pulse width of 100 fsec and the uv-enhanced microchannel plate detector.
  • Fluorescence quantum yield was determined by absorbance and fluorescence measurements of the photoproduct referenced to a known standard, 2aminopurine (2AP) , which has a quantum yield of 68% in water with pH 7 phosphate buffer, and 95% in unbuffered water.
  • 2AP 2aminopurine
  • Figs. 3 (a) -3(f) show the absorption spectra before and after uv illumination, and the fluorescence emission and excitation spectra taken after uv illumination, for various dGMP concentrations in an unbuffered solution of 30% glycerol in water. Illumination conditions were 50 mW of 275 nm light. The absorption measurements show the characteristic decrease in the parent (dGMP) absorption with the appearance of a new feature, with maxima at 305 nm and 220 nm. Clean isosbestic points can be seen.
  • the fluorescence emission essentially zero before illumination, shows bright fluorescence after illumination, with an emission maximum of 365 nm, and excitation maxima at 220 nm, 248 nm, and 303 nm.
  • the excitation spectrum agrees with the photoproduct absorption features.
  • the main difference between the three concentrations is the relative photoconversion yields.
  • 2-aminopurine (2AP) has an extinction coefficient 7150 M _1 cm “1 at 303 nm. Considering the uv energy absorbed (7.4 Joules), and the number of molecules converted (8 uM) , the photochemical quantum yield was 0.12%.
  • the emission of the 2-propanol product contains a hump in the emission spectra in the 380-450 nm range, suggesting more than one emission maximum and hence a second red-shifted photoproduct.
  • Photochemistry using xanthosine 5 ' -monophosphate (XMP) under similar conditions yielded a fluorescent photoproduct, with a photochemical yield smaller than dGMP, and with fluorescence excitation and emission spectra red-shifted (excitation maximum 315 nm, emission maximum 375 nm) from that of the dGMP-photoproduct.
  • the fluorescence properties appear similar to isoinosine, a fluorescent analog of inosine, and when taken together with the similarity between the dGMP photoproduct and 2-aminopurine, suggests that photochemical modification of the parent nucleotide may involve changes at the C(6) position of the base ring structure.
  • the nucleotide dAMP exhibited a large photochemical rate in glycerol/water, ethylene glycol/water, and 2-propanol/water, and produced a photoproduct with about 50-times less fluorescence than the dGMP photoproduct.
  • the excitation spectra peaked at 315 nm, while the emission peaked at 410 nm. The less intense emission, and the fact that the excitation spectra was red-shifted from the photoproduct absorption peaks, suggests that the dAMP-photoproduct fluorescence contains a minor fluorescent component such as a tautomer.
  • Spectroscopy of the dGMP-photoproduct was performed using dilute solutions of the photolyzed samples described above.
  • the emission and excitation spectra of a solution containing 7.7 nM dGMP-photoproduct in unbuffered water is shown in Fig. 6(a).
  • fluorescence from a solution of 2-aminopurine, adjusted in concentration to have the same absorbance (5xl0 ⁇ 4 ) at 303 nm as the photoproduct solution is shown in Fig. 6(b).
  • the photoproduct spectra are nearly identical to the 2AP-spectra, with a small blue-shift of the excitation and emission maxima (emission wavelength of 265 nm vs. 269 nm for 2AP) .
  • a quantitative measurement of the quantum yield of the photoproduct is 40%, based on comparison to the fluorescence intensity of 2AP (quantum yield 95% in water) .
  • the fluorescence lifetime for optically thin, unbuffered solutions of dGMP-photoproduct and 2AP are shown in Fig. 7.
  • the lifetimes are 7.5 nsec and 9.5 nsec, respectively. Due to the similarity of the absorption and emission spectra between dGMP-photoproduct and 2AP, as well as the approximate equality of the extinction coefficients, the single- exponential lifetime for dGMP-photoproduct (7.5 nsec) suggests its quantum yield should be about 65%, based on Strickler-Berg arguments.
  • the pH dependence of the fluorescence intensity of the guanosine photoproduct is shown in Fig. 8. (The dGMP-product behaves similarly) . As can be seen, the emission is approximately constant from pH 4.5 to 11, with some variation probably arising from the type of buffer used. The emission intensity drops to half maximum at pH 3.5 and 11.5. This dependence is in close agreement with that found by Ward for 2AP-riboside monophosphate, who reported excited-state pK values of 3.6 and 12.1. Ward,D. C. , and Reich, E. , "Fluorescence Studies of Nucleotides and Polynucleotides. I.
  • a simple procedure was used to make substrates with nucleotides bound to the surface.
  • a quartz substrate one surface of which was coated by vacuum deposition with a thin, 10-nm aluminum-oxide film, is covered with an aqueous solution containing nucleotides at a concentration of typically 50-200 nM. After a few minutes to allow the nucleotides in solution to diffuse to the surface, the quartz substrate is rinsed with water to remove any nucleotides not bound to the surface, then dried with nitrogen, and covered with either water or another solvent.
  • nucleotide binding efficiency to a quartz surface with no alumina is down about 10, 000-fold.
  • alumina-bound nucleotides can remain bound for hours, with a 1/e off-rate of about 1/(7 hrs) at room temperature.
  • the use of a buffered solution of nucleotides may or may not inhibit binding, e.g., a basic glycine buffer reduces binding on alumina by greater than 10-fold, whereas a basic Tris buffer does not. But once the nucleotides are bound to the surface, changing the solution buffer does not appear to displace the nucleotides from the surface, although very acidic solutions or phosphate-buffered solutions appear to displace bound nucleotides.
  • Fig. 10(a) shows the change in fluorescence intensity with time from fluorescein-labeled dUTP bound to an alumina surface. Low excitation power is used to minimize bleaching of the fluorophore.
  • the decay in the emission with time reflects the reduction in the number of nucleotides bound to the surface.
  • the data can be fit assuming an exponential decay of the number of nucleotides bound.
  • the fitted off-rate was determined to be 3.8 x 10 ⁇ 5 sec "1 , which yields a 1/e off time of about 7 hours. Similar off times were found for monophosphate nucleotides.
  • FRAP Fluorescence Recovery After Photobleaching
  • Fig. 11 shows the fluorescence lifetime of surface-bound 2-aminopurine triphosphate (2APTP) and the bound nucleotides from a solution containing dGMP-photoproduct (taken from the photolyszed bulk solution of Fig. 3(e)).
  • the lifetimes were measured using unbuffered water as the solution in contact with the surface-bound nucleotides.
  • the lifetimes are best fit to a two-component decay, as opposed to the lifetimes measured in solution, which yielded single-exponential decays.
  • the slow component (6-7 nsec) is approximately equal to that expected based on the solution decay, while about 16% of the bound nucleotides exhibit a short lifetime of about 1.5 nsec.
  • the fluorescence decay can be fit well with a single exponential, and we estimate that the fraction of bound nucleotides with a short-lifetime decay is reduced to less than 5%.
  • various methods to further enhance homogeneous binding sites can be employed, such as capping the acidic sites prior to nucleotide binding. Langmuir-Blodgett films may also be used to provide more homogeneous binding. The conclusion drawn from these lifetime measurements are that the quantum yield for nucleotides on an alumina surface is essentially that found in solution, while a small portion (5-15%) of the bound nucleotides have a quantum yield about 4x smaller than that in solution.
  • the ultimate measure of detection sensitivity is the detection of uv-excited, uv-fluorescent single molecules.
  • key issue is the reduction of background fluorescence from the medium surrounding the single molecule of interest.
  • low background fluorescence is achieved by the combined use of (1) high- purity quartz substrates (Corning 7904, polished to better than 5-10 scratch-dig and sold by CVI, Inc.), (2) very thin films of alumina (aluminum oxide will fluoresce under uv illumination, so only a minimum film thickness to insure coverage of the quartz substrate is necessary) , cleaned in a hot acid/peroxide bath prior to use for immobilizing a molecule, (3) evanescent-wave excitation so that only a region within about 0.03 microns of the alumina/quartz surface is excited, and so the solvent is minimally excited, (4) ultrapure water and solvents free of fluorescent impurities, (5) a lOOx, oil immersion quartz objective, the solid angle of which collects a
  • the following method was used to detect single dGMP- photoproduct nucleotides.
  • An alumina-coated quartz substrate was first coated with dGMP photoproduct at a coverage of about 1000 molecules per square micron. This sample was then placed in a glove bag, covered with a drop of unbuffered water, and purged with nitrogen for 30 minutes. While still in the nitrogen-filled glove bag, the sample was then covered with a quartz coverslip, using a thin elastomer (PDMS) to seal the coverslip on the alumina/quartz substrate. In this way, we could make a sealed cartridge containing nitrogen- purged water (water thickness about 20 microns) overcoating the nucleotides bound to the alumina/quartz substrate.
  • PDMS thin elastomer
  • This "sandwich"-like cartridge was then placed on the microscope stage for evanescent excitation at 293 nm, with collection by an oil-immersion, lOOx objective (1.2 NA) through the coverslip.
  • lOOx objective 1.2 NA
  • the fluorescence from the sample surface was imaged onto a CCD camera.
  • each camera pixel size of 25 microns
  • each camera pixel records fluorescence from an average of about 67 molecules.
  • a small region on the alumina/quartz was first illuminated with high intensity uv light (293 nm) , to bleach all of the nucleotides in that region.
  • the uv intensity was then turned down, and fluorescence images taken of the sample. Since the water solution above the surface will always contain a very small concentration of photoproduct nucleotides that have desorbed from other unbleached regions of the surface, these nucleotides will continually re-bind to the surface, and hence some will rebind to the bleached region. Therefore, a fluorescence image of this bleached region of the sample can show single nucleotides binding to the alumina surface at random locations.
  • Fig. 12(a) shows one series of fluorescent images taken with 0.5 mWatts of 293 nm power (an intensity of about 50
  • Each image, or frame collects light for 2 sec, and there is a dead time between frames of about 1/2 sec during which the data is read-out and the camera is off.
  • These images show that single bright pixels with signals well above the noise appear for typically one to several frames, and then disappear.
  • Fig. 12(b) we show a linecut through the images in Fig. 12(a) for several frames, to shown more clearly the appearance and disappearance of single nucleotide molecules. We believe these bright pixels represent single photoproduct nucleotides binding to the alumina, which after a brief period, undergo bleaching.
  • dGMP was deposited on an alumina-film/quartz substrate, as described above, to an approximate surface coverage of about 10 4 /um 2 .
  • a 30% glycerol/water drop was placed over the surface, and the whole sample area purged with nitrogen for 30 min. The sample was then irradiated with uv light. Based on the spectrum and intensity of the fluorescence in the bandwidth of the photoproduct under 260 nm illumination, these measurements indicated about 10% of the parent dGMP nucleotide was converted to a fluorescent product.
  • the 260 nm light both initiates the photochemistry, and is used to excite the photoproduct fluorescence.
  • the alumina/quartz substrate was replaced with just quartz, and covered with a drop of 30 uM dGMP in water.
  • significant photoproduct was observed after purging this arrangement with nitrogen, indicating that the photochemical reaction proceeds very close (within 10 nm) to the surface.
  • the lower photoproduct conversion may arise in the molecular interaction between nucleotide and reagent.
  • molecular orientation of the bound nucleotide may hinder the necessary approach by the reagent molecules, thus preventing a necessary orientation and proximity.
  • This can be tested using a less rigid (more fluid-like, but equally strong) binding layer, to allow the nucleotides greater rotation or conformational freedom. Additional information on conversion yield for alumina are under way, with initial tests to understand the role of solvent pH on the photochemistry.
  • the solution photochemistry described above requires an reagent that can contact the nucleotide, and we have demonstrated examples of such a reagent in glycerol, isopropanol, and ethylene glycol.
  • the active reagent cannot just be water, since photolysis of a solution containing only water and dGMP does not result in a fluorescent product.
  • the photochemical yield is very low. So an active component of the photochemistry is the replacement of dissolved oxygen with another gas such as nitrogen or argon.
  • photochemical reactions precede from the triplet state of a molecule.
  • a nucleotide absorbs light and as a result occasionally forms a triplet state, a state that is chemically reactive and usually metastable (lifetime from 10 usec to 1 msec in solution) .
  • a long-lived triplet state is desirable to increase the time for encounters with reagent molecules. This accounts for our need to purge the solution of dissolved oxygen, since oxygen is a well-known triplet quencher that shortens the triplet lifetime.
  • Other methods to modify the triplet state dynamics for example, by increasing the intersystem-crossing rate, should improve the photochemical quantum yield.
  • photochemistry from reagents to products often takes place via radical intermediates, formed initially by hydrogen- or electron- abstraction from a donor reagent molecule.
  • the alcohols we have used in our work are known to be good hydrogen-atom donors.
  • Useful reagents for photochemistry could be chosen from a list of hydrogen-atom donating molecules that includes alcohol-, sugar-, amine-, hydrocarbon- containing compounds, and this list includes nucleic acid components as well. Since the photochemistry may proceed where an active reagent absorbs the uv light, followed by the abstraction of a hydrogen atom from nucleotide, reagents that are good hydrogen-atom acceptors, such as aromatic ketones like benzophenone, may also be helpful.
  • the principal issue in the choice of the reagent is the specificity of the photochemical reaction to produce a fluorescent product of a nucleotide, while additionally that product should not undergo photochemical reactions itself.
  • the photochemical reaction can be obtained with uv- illumination at a wavelength outside of the absorption band of the nucleotide by use of a photosensitizer.
  • the reaction solution would consist of nucleotides, active reagent and sensitizer.
  • Ultraviolet light would be absorbed by the sensitizer, which either directly formed radicals of the reagent or nucleotides, or by energy transfer excited the nucleotide triplet state, which then formed radicals.
  • Sensitizers are well-known in photochemistry, an example of which is acetone.
  • nucleotides immobilized on a surface we used one simple method of binding that involved the electrostatic attraction between the negative phosphate of the nucleotide and the positive ion of a metal oxide, alumina. Other surfaces may be used with equally good binding properties, but which also allow the nucleotides greater freedom of motion.
  • a metal- ion ligand connected by a linker that is covalently attached to a substrate surface such as quartz could provide a suitable binding site for nucleotides.
  • the length of the linker should be chosen to allow the bound nucleotide rotational and conformational motion, which is desirable to increase the photochemical reaction rate.
  • the binding is not limited to electrostatic forces due to metal ions.
  • the nucleotides could be immobilized on a surface by application of an electric field, either externally applied or due to charged particles co-located on a surface.
  • Another advantageous binding arrangement would be the use of enzymes or proteins covalently attached to a surface, that either generally or specifically bind nucleotides with high affinity.
  • Any binding layer in all cases would likely be as thin as practical, but does not need to be a continuous film, and may intentionally or unintentionally consist of strips or islands of binding material, so chosen to advantageously minimize diffusion of the nucleotides.
  • the thin binding layer on a substrate used to immobilize the nucleotides may also act itself as a reactive solvent, or otherwise participate in the photochemistry.
  • the substrate containing the surface-immobilized nucleotides can be processed to further decrease the off-rate and limit diffusion of the bound nucleotides.
  • a solution containing "blocking" molecules that bind to the alumina can be spread over the surface.
  • a blocking molecule could be riboside-monophosphate (a nucleotide without a base) that binds up the remaining available sites on the alumina surface, without displacing the nucleotides.
  • the blocking molecules act to (1) prevent binding of reagent molecules in subsequent processing steps, and (2) further reduce the diffusion of nucleotides along the surface (by analogy to the restricted movement of cars in a filled parking lot) .
  • this solution can be washed off, and replaced with another.
  • the sample is then ready for the process of fluorescence enhancement and detection of the surface bound nucleotides.
  • a non-polar solvent can be placed over the binding surface, which we have found can reduce the nucleotide off rate five-fold. Additionally the substrate can be stored at low temperature indefinitely, with no displacement of the nucleotides, until needed for the detection process.
  • the polarization of the light can be changed during the course of the photochemistry, or the sample containing the nucleotides reoriented, for the purpose of providing equal amounts of light energy along the three possible directions in space, insuring that every molecule receives equal excitation, independent of its orientation.
  • the geometry for the photochemistry of the nucleotides can be arranged so that either a real (i.e. propagating) uv electric field or an evanescent (i.e. exponentially-damped, non- propagating) uv electric field is used in the photochemistry.
  • an evanescent field can be advantageously used to excite only the nucleotides and/or the reactive solvent very close to the nucleotides.
  • confocal single channel approach One advantage of the confocal single channel approach is that the fluorescence decay at each spot on the sample can be analyzed in software to yield the presence and identity of a nucleotide based on fluorescence decay time, rather than just the fluorescence intensity. For this arrangement, the sample is rastered so that all spots on the sample are probed. This detection scheme can be extended to incorporate a slit confocal geometry, which is intermediate between our full-field geometry and a single point confocal geometry.

Abstract

La présente invention concerne un procédé de fabrication de photoproduits fluorescents de nucléotides utilisant certains solvants, dans des solutions dégazées au cours d'une radiation par ultraviolets. Ces photoproduits peuvent être hautement fluorescents (rendement quantique QY de l'ordre de 50%) dans des solutions aqueuses à température ambiante et dans une large plage de pH et présentent une faible probabilité de blanchissement de l'ordre de 10-4 avec une amélioration anticipée à l'aide d'agents d'extinction à triplet. Ces nucléotides modifiés lorsqu'ils sont liés et immobilisés sur une surface d'aluminium peuvent être détectés au niveau de la simple molécule. Leur intérêt réside dans l'amélioration significative de la détectabilité desdits nucléotides par fluorescence et dans le fait qu'ils peuvent offrir un ensemble attractif de fluophores par application au séquençage d'ADN base/temps.
PCT/US1998/018817 1997-09-11 1998-09-10 Procede de fabrication de photoproduits de nucleotides fluorescents pour sequençage d'adn et analyse WO1999013110A1 (fr)

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US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
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WO2001016371A2 (fr) * 1999-08-27 2001-03-08 Gen-Probe Incorporated Procede d'identification de phosphoramidites a une base d'acide nucleique par la spectroscopie a fluorescence
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US9657344B2 (en) 2003-11-12 2017-05-23 Fluidigm Corporation Short cycle methods for sequencing polynucleotides
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
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US9539571B2 (en) 2010-01-20 2017-01-10 Honeywell International Inc. Method to increase detection efficiency of real time PCR microarray by quartz material
WO2011088588A1 (fr) * 2010-01-20 2011-07-28 Honeywell International, Inc. Réacteur utilisable en vue d'une analyse quantitative d'acides nucléiques
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