WO2001016371A2 - Procede d'identification de phosphoramidites a une base d'acide nucleique par la spectroscopie a fluorescence - Google Patents

Procede d'identification de phosphoramidites a une base d'acide nucleique par la spectroscopie a fluorescence Download PDF

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Publication number
WO2001016371A2
WO2001016371A2 PCT/US2000/023422 US0023422W WO0116371A2 WO 2001016371 A2 WO2001016371 A2 WO 2001016371A2 US 0023422 W US0023422 W US 0023422W WO 0116371 A2 WO0116371 A2 WO 0116371A2
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nucleic acid
sample
fluorescent
phosphoramidite
excitation
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PCT/US2000/023422
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WO2001016371A3 (fr
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Matthew C. Friedenberg
Michael M. Becker
Mehrdad Majlessi
James Russell
William G. Weisburg
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Gen-Probe Incorporated
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Publication of WO2001016371A3 publication Critical patent/WO2001016371A3/fr

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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

Definitions

  • This invention relates to synthesis of oligonucleotides, and specifically relates to a method for monitoring coupling reactions during oligonucleotide synthesis by detecting the presence of nucleic acid base phosphoramidites in the effluent of a synthesis reaction device.
  • nucleic acid o gomers such as oligonucleotides or oligonucleotide analogues
  • oligonucleotides involve the step-by-step coupling of individual nucleic acid base phosphoramidites to a growing chain that is attached by the 3'-nucleos ⁇ de to a solid support (Beaucage et al., 1982, Tetrahedron Lett 22(20):1859-1862, Beaucage et al., 1992, Tetrahedron 48(12).2223-2311 )
  • the solid phase may be controlled pore glass (CPG) or chemically inert, hydrophobic membranes.
  • a nucleic acid ⁇ -cyanoethyl phosphoramidite monomer containing one of the common bases (A, T, G, C or I) or an abasic monomer is provided for each coupling step, which typically involves a cycle of five chemical reactions (detritylation or deblocking, activation, coupling, capping and oxidation)
  • the sequence of addition of the phosphoramidite monomers corresponds to the sequence of bases desired in the nucleic acid oligomer product.
  • a mixture of two or more nucleic acid phosphoramidite monomers is provided for the individual coupling step that corresponds to the residue for which redundancy is desired
  • the oligomer is cleaved from the support and protecting groups are removed. Determining the accuracy of nucleic acid oligomer synthesis is often crucial before the produced oligomer can be used, for example, as a therapeutic or diagnostic reagent
  • the composition and sequence of a nucleic acid oligomer can be determined by analysis independent of synthesis (e.g , by sequencing the oligomer product).
  • sequencing provides limited information on the accuracy of coupling during synthesis because more than one base will be present for a given residue, thus providing results that resemble sequencing artifacts.
  • some residues in a synthetic oligomer e.g., modified bases or abasic sites
  • sequencing the product or otherwise performing separate analysis procedures to verify the accuracy of a nucleic acid oligomer synthesis is both costly and time-consuming. The costs include analysis costs, consumption of a portion of the product, and resynthesis costs where a product must be rejected because of inaccuracies in the sequence which are only detecte ⁇ after the product has been completely made.
  • Fluorescence spectroscopy is a well known analytical tool for detecting a variety of compounds (Stryer, 1978, Ann. Rev. Biochem. 47.819-846). Fluorescence has also been used to detect DNA fragments generated during gene sequencing. These methods, however, generally rely on addition of fluorophores during the sequencing reactions (Smith et al., 1986, Nature 321 :674-679; Ansorge et al , 1987, Nucleic Acids Res. 15(11 J.4593-4602, Prober et al , 1987, Science 238:336-341 )
  • Fluorescence is one analytical technique that has been used to analyze nucleic acid base composition
  • Nucleic acid bases (A, T, G and C)
  • Nucleic acid bases are only weakly fluorescent using excitation wavelength ranging from 250 to 300 nm and emission wavelengths ranging from 300 to 330 nm (Cantor, et al., Biophysical Chemistry, Part II Techniques for the Study of Biological Structures and Function (W H Freeman & Co., San Francisco, 1980), Table 8-2 at page 443).
  • the level of fluorescence of the bases in this spectral region requires concentrations of bases that are generally higher than those used in synthetic reactions. That is, the concentrations of phosphoramidites used during nucleic acid oligomer synthesis generally does not permit detection of fluorescence resulting from the base.
  • This invention detects the individual fluorescent spectra of nucleic acid base phosphoramidites, resulting from the combination of a protecting group and a base, to monitor the efficiency of nucleic acid synthesis directly from the effluent of a synthesis reaction.
  • a method for detecting and identifying a nucleic acid base phosphoramidite in a sample solution includes the steps of providing a sample containing at least one nucleic acid base phosphoramidite in solution, illuminating the sample over a range of excitation wavelengths, then measuring fluorescent emission intensity of the sample over the range of excitation wavelengths, thereby generating a fluorescent excitation spectrum of the sample, comparing the fluorescent excitation spectrum of the sample with a fluorescent excitation spectrum of at least one known nucleic acid phosphoramidite, and determining that the fluorescent excitation spectrum of the sample matches one or more features of the fluorescent excitation spectrum of the known nucleic acid phosphoramidite, thereby indicating that the sample contains a nucleic acid phosphoramidite identical to a known nucleic acid phosphoramidite.
  • the step of measuring fluorescent emission intensity uses a fixed emission wavelength in a range having a lower limit of about 250 nm to about 400 nm and an upper limit of about 400 nm to about 650 nm.
  • the measuring step preferably uses a range of excitation wavelengths having a lower limit of about 250 nm to about 350 nm and an upper limit of about 400 nm to about 550 nm.
  • the range of excitation wavelengths is from about 275 nm to about 400 nm, and more preferably from about 310 nm to about 377 nm.
  • the measuring step is performed over a range of excitation wavelengths of about ⁇ 40 nm surrounding a peak location of a known nucleic acid base phosphoramidite, whereas in another embodiment the range is about ⁇ 25 nm surrounding a peak location of a known nucleic acid base phosphoramidite
  • the features of a fluorescent excitation spectrum used in comparing spectra and determining matches include peak location, peak intensity, line shape or a combination thereof
  • generating the fluorescent excitation spectrum is completed in about 1 second or less.
  • the method may be practiced in a system that includes a nucleic acid synthesizer operably connected to a fluorescence detector
  • the fluorescence detector is triggered by the nucleic acid synthesizer such that a fluorescent excitation spectrum is measured for each of the samples associated with a coupling reaction of a nucleic acid synthesis, such as, for example, an influent solution used in the synthesis, an effluent solution from a synthetic step or a reaction mixture within the synthesizer.
  • the system further includes an automated means for determining identity of a nucleic acid base phosphoramidite is present in the sample, such as a computer that uses an algorithm for comparing the fluorescent excitation spectra of a given sample and known phosphoramidites. More preferably, the system is capable of both identifying and quantifying the nucleic acid base phosphoramidite present in the sample.
  • One embodiment of the method is used to determine the accuracy of nucleic acid synthesis, in which, in the providing step, the sample is an influent solution to, an effluent solution from, or a solution within a synthetic nucleic acid coupling reaction chamber; the illuminating step uses fluorescent excitation wavelengths in a range of about 200 nm to about 500 nm; the measuring step measures fluorescent emission intensity of the sample at a fixed wavelength over the range of excitation wavelengths; and the determining step identifies the nucleic acid base phosphoramidite present in the sample based on its features that match features of a fluorescent excitation spectrum of a known nucleic acid phosphoramidite, thereby indicating a nucleic acid base added during a synthetic nucleic acid coupling reaction.
  • the measuring step uses a fixed wavelength between about 250 nm and about 650 nm
  • Another embodiment is a system of determining the accuracy of nucleic acid synthesis according to the above-described method, wherein the system comprises a nucleic acid synthesizer operably connected to a fluorescence detector
  • Another aspect of the invention is a method for detecting and identifying a nucleic acid base phosphoramidite in a sample solution. This method includes the steps of providing a sample containing at least one nucleic acid base phosphoramidite in solution, illuminating the sample at a fixed excitation wavelength, and measuring fluorescent emission intensity of the sample over a range of emission wavelengths, thereby generating a fluorescent emission spectrum of the sample.
  • the method includes comparing the fluorescent emission spectrum of the sample with a fluorescent emission spectrum of a known nucleic acid phosphoramidite, and determining that the fluorescent emission spectrum of the sample matches one or more features of the fluorescent emission spectrum of the known nucleic acid phosphoramidite, thereby indicating that the sample contains a nucleic acid phosphoramidite identical to the known nucleic acid phosphoramidite.
  • the fixed excitation wavelength is in a range having a lower limit from about 250 nm to about 350 nm and an upper limit of about 350 nm to about 460 nm, more preferably in a range from about 300 nm to about 380 nm, and most preferably is in a range from about 310 nm to about 377 nm.
  • the measuring step preferably uses emission wavelengths in a range having a lower limit of about 250 nm to about 350 nm and an upper limit of about 400 nm to about 650 nm, and more preferably uses emission wavelengths in a range from about 300 nm to about 500 nm.
  • the features of a fluorescent emission spectrum include peak location, peak intensity, line shape or a combination thereof, which are used in determining matches between compared spectra
  • a preferred embodiment of the invention further adds the steps of illuminating the sample over a range of excitation wavelengths, then measuring fluorescent emission intensity of the sample over the range of excitation wavelengths, thereby generating a fluorescent excitation spectrum of the sample, comparing the fluorescent excitation spectrum of the sample with a fluorescent excitation spectrum of at least one known nucleic acid phosphoramidite, and determining that one or more features of the fluorescent excitation spectrum of the sample matches one or more features of a fluorescent excitation spectrum of a known nucleic acid phosphoramidite, thereby indicating that the sample contains a nucleic acid phosphoramidite identical to a known nucleic acid phosphoramidite.
  • the steps of generating a fluorescent excitation spectrum and generating a fluorescent emission spectrum are performed substantially simultaneously.
  • This method that includes generating both a fluorescent excitation spectrum and a fluorescent emission spectrum is particularly useful for resolving individual components of a sample solution that contains a mixture of phosphoramidites.
  • the method is used to determine the accuracy of nucleic acid synthesis, in which, in the providing step, the sample is an influent solution to, an effluent solution from, or a solution contained within a synthetic nucleic acid coupling reaction chamber, the measuring step measures fluorescent emission intensity of the sample over a range of emission wavelengths between about 250 nm and about 650 nm; and the determining step identifies the nucleic acid base phosphoramidite present in the sample based on a match of one or more features of the spectrum of the sample and the spectrum of a known nucleic acid phosphoramidite, thereby indicating a nucleic acid base added during a synthetic nucleic acid coupling reaction
  • Another aspect of the invention is a method for detecting and identifying a nucleic acid base phosphoramidite in a sample solution This method includes providing a sample containing at least one nucleic acid base phosphoramidite in solution and illuminating the sample with at least two fixed fluorescent excitation wavelengths.
  • the method includes measuring fluorescent emission intensity of the sample at the fixed excitation wavelengths, determining a specific fluorescent excitation wavelength at which maximal fluorescent emission was measured for the sample and comparing the specific fluorescent excitation wavelength at which maximal fluorescent emission was measured for the sample with relative intensities of fluorescent emissions for known nucleic acid base phosphoramidites at the fixed fluorescent excitation wavelengths.
  • the method then includes identifying the nucleic acid base phosphoramidite present in the sample based on its maximal fluorescent emission which occurs at substantially the same fixed excitation wavelength that results in maximal fluorescent emission of a known phosphoramidite.
  • the illuminating step uses at least four fixed fluorescent excitation wavelengths, which preferably are about 313 nm, about 333 nm, about 365 nm and about 377 nm.
  • the sample provided is an influent solution to a synthetic nucleic acid coupling reaction, an effluent solution from a synthetic nucleic acid coupling reaction, or a solution contained within a coupling reaction chamber of a nucleic acid synthesizer.
  • 1 is a graph showing the normalized excitation spectra ("Fluorescent Units”), after subtraction of background, over a range of 280 nm to 460 nm for: abasic phosphoramidite ("Abasic"), ph- rU having a tBDMSi protecting group (“rU”), ph-dT (“dT”), ph-dG-iBu (“dG iBu”), ph-dC-Ac (“dC Ac”), ph- rC (“rC”), ph-dA-Bz ("dA Bz”), ph-dC-Bz (“dC Bz”) and ph-rC-Bz ("rC Bz”) at 0.07 M in acetonitnle.
  • Abasic abasic phosphoramidite
  • rU ph- rU having a tBDMSi protecting group
  • dT ph-dT
  • FIG. 2 is a graph showing the normalized emission spectra ("Fluorescent Units”), after subtraction of background, over a range of 300 nm to 600 nm for: abasic phosphoramidite ("Abasic”), ph- rU having a tBDMSi protecting group (“rU”), ph-dT (“dT”), ph-dG-iBu (“dG iBu”), ph-dC-Ac (“dC Ac”), ph- rC ( ⁇ C”), ph-dA-Bz ("dA Bz”), ph-dC-Bz (“dC Bz”) and ph-rC-Bz ("rC Bz”) at 0.07 M in acetonitnle.
  • Abasic abasic phosphoramidite
  • rU ph- rU having a tBDMSi protecting group
  • dT ph-dT
  • dG iBu
  • FIG. 3 is a graph showing the normalized excitation spectra ("Fluorescent Units”), after subtraction of background, over a range of 250 nm to 450 nm for: abasic phosphoramidite ("Abasic”), ph- rU having a tBDMSi protecting group (“rU”), ph-dT (“dT”), ph-dG-iBu (“dG iBu”), ph-dC-Ac (“dC Ac”), ph- rC (“rC”), ph-dA-Bz ("dA Bz”), ph-dC-Bz ('dC Bz”) and ph-rC-Bz ("rC Bz”) at 0.007 M in acetonitnle FIG.
  • Abasic abasic phosphoramidite
  • rU ph- rU having a tBDMSi protecting group
  • dT ph-dT
  • FIG. 4 is a graph showing the normalized emission spectra ("Fluorescent Units”), after subtraction of background, over a range of 300 nm to 600 nm for: abasic phosphoramidite ("Abasic”), ph- rU having a tBDMSi protecting group (“rU”), ph-dT (“dT”), ph-dG-iBu (“dG iBu”), ph-dC-Ac (“dC Ac”), ph- rC (“rC”), ph-dA-Bz (“dA Bz”), ph-dC-Bz (“dC Bz”) and ph-rC-Bz ("rC Bz”) at 0.007 M in acetonitnle
  • FIG 5 is a graph showing the relative intensity of fluorescent emission detected at 460 nm for effluent from a DNA synthesis reaction during a 1.0 mm washing interval following a synthetic coupling step adding a dC base, where the excitation wavelength was 375 nm.
  • FIG 6 is a graph showing excitation spectra over a range of 275 nm to over 400 nm for phosphoramidites ph-dT (— A — ). ph-dC-Bz (—
  • FIG. 7 is a graph showing excitation spectra obtained over 275 nm to 400 nm for ph-dC-Bz at different times ( A ,0.15 m ⁇ n; ⁇ , 0.2 mm; 12, 0.25 mm; ⁇ , 0.3 mm; *, 0.35 mm; and* , 0.4 mm) after the effluent exited the synthesizer.
  • FIG. 8A to 8H show the individual excitation spectra (measured in mv, shown on the Y-axis) over excitation wavelengths of 275 nm to 400 nm (shown on the X-axis) obtained for sequential effluent samples obtained during synthesis of a 9-mer oligonucleotide having the sequence 5' AACGGTTCT 3', starting from the C at base 2, adjacent to the 3' T residue (FIG. 8A), and proceeding to base 9, the 5' A residue of the oligonucleotide (FIG. 8H).
  • DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the relevant art
  • Nucleic acid base phosphoramidite means a compound comprising a ⁇ bose or deoxynbose sugar joined to a punne or pynmidme base, or analog thereof, or no base (an “abasic phosphoramidite”), at least one protecting group attached to the compound, and a phosphoramidite moiety attached to the sugar moiety
  • Fluorescent excitation spectrum means a quantitative measurement of fluorescent emissions detected at a single wavelength for a compound illuminated over a continuous range of fluorescent excitation wavelengths
  • Fluorescent emission spectrum refers to a quantitative measurement of fluorescent emissions detected over a continuous range of fluorescent emission wavelengths for a compound illuminated at a single fluorescent excitation wavelength
  • Peak location is a feature of a fluorescent spectrum that is the wavelength at which a relatively high fluorescent emission is detected for a compound illuminated with one or more fluorescent excitation wavelengths A compound may exhibit one or more peaks at different locations over a range of wavelengths
  • Peak intensity is a feature of a fluorescent spectrum that is the quantitative measurement of fluorescent emission that is detected for a compound illuminated with one or more fluorescent excitation wavelengths
  • Line shape is a feature of a fluorescent spectrum that refers to the overall appearance of a graphic display of a fluorescent spectrum, including the locat ⁇ on(s), relative he ⁇ ght(s) and broadness of peak(s) or valley(s)
  • Substantially simultaneously refers to generating a fluorescent spectrum using a series of different excitation and emission wavelengths over a range to scan a single sample That is, each data point represents the results of a different emission and excitation wavelength relative the other data points that make up the entire spectrum
  • Maximum fluorescent emission means the greatest amount of fluorescent emission detected for a single sample following illumination with at least two different fluorescent excitation wavelengths
  • Fiberd fluorescent wavelength means a discrete wavelength or point on a wavelength spectrum capable of causing fluorescent emission or at which a fluorescent emission is detected
  • Fluid solution means a solution containing at least one compound that flows into a device or during a process (i.e., a fluid input).
  • effluent means a solution containing at least one compound that flows out of a device or during a process (i.e., a fluid output).
  • An “effluent wash” is a washing solution that flows out of a device or following a washing step in a process.
  • Associated with a coupling reaction refers to a solution or compound that is fluid input for a coupling step in nucleic acid synthesis, fluid output following a nucleic acid synthesis coupling step, or a solution containing at least one reaction component of a nucleic acid synthesis coupling step which is in a reaction chamber or device for nucleic acid synthesis. It will be appreciated that on some synthesis devices, a solution may cycle through a reaction chamber more than once before being an effluent solution that exits the reaction chamber for the last time.
  • sample means a solution or portion of a solution that potentially contains at least one nucleic acid base phosphoramidite.
  • Automated means that at least one step in a method is controlled by the operation of a mechanical and/or electronic device that takes the place of a human operator or practitioner.
  • nucleic acid oligomers particularly oligonucleotides and oligonucleotide analogues
  • an excess of a specific phosphoramidite is used in each coupling step that adds a new base (or abasic sugar-phosphate ) to the oligomenc chain (Beaucage et al., 1982, Tetrahedron Lett.
  • the effluent from a synthesis device typically contains unreacted phosphoramidite monomers
  • a synthesis device e.g , an automated DNA synthesizer
  • phosphoramidites unless otherwise defined, are herein referred to by the abbreviations ph-dA, ph-dT, ph-dG and ph-dC, for deoxy ⁇ bose phosphoramidites having the A, T, G and C bases, respectively, and ph-rA, ph-rU, ph-rG and ph-rC for the ribose phosphoramidites having the A, U, G and C bases, respectively.
  • Protecting groups on the phosphoramidite compounds are generally referred to by their commonly accepted abbreviations (e.g., Bz and iBu), which are appended to the above abbreviations (e.g., ph-dG-iBu).
  • the fluorescent excitation and emission spectra of the various phosphoramidites were used to identify the phosphoramidite used in a nucleic acid synthesis step, thereby identifying the individual bases added at sequential steps during nucleic acid synthesis.
  • the excitation spectra are linear representations of normalized fluorescent intensity measured over a range of excitation wavelengths
  • the emission spectra are linear representations of normalized fluorescent intensity measured over a range of emission wavelengths.
  • each individual phosphoramidite compound in solution can be detected and identified based on its spectral features, thus allowing detection and identification of the nucleic acid base phosphoramidite used in each synthetic coupling step by monitoring a solution associated with each coupling reaction (e g., effluent wash).
  • a solution associated with each coupling reaction e g., effluent wash.
  • an effluent fluorescent spectrum is used to monitor the compound added during each coupling step of a synthesis.
  • the effluent of each coupling step may be monitored after completion of a synthesis, but that would not provide information on the accuracy and efficiency of coupling steps during synthesis, when a synthesis could be terminated or adjusted if coupling was inadequate or an error occurred
  • this method is advantageous because it can be automated, partially or completely, to allow a practitioner or automated system to monitor the accuracy of a synthesis substantially simultaneous with the synthetic reactions
  • the method may be practiced as part of an automated synthesis system by attaching any of a variety of well known fluorescent detector systems to an influent line, an effluent line or a reaction chamber of a nucleic acid synthesis reactor, thereby monitoring the incoming chemicals, the residual excess chemicals or chemicals present in a coupling reaction, respectively, for each synthetic coupling
  • the fluorescent detection system may be used to monitor the presence of chemicals in the synthetic reaction chamber by using a fluorescent transparent material to
  • effluent samples can be collected in tubes or wells of a multi-well plate following each coupling step and later monitored in an automated detection system.
  • Fluorescent detectors are well known in the art (e.g., see Green et al , 1986, J. Chromatography 352:337-343) and many are commercially available The present invention does not rely on any particular detector system
  • Different phosphoramidites have different excitation and emission spectra that generally result from a fluorescent dimethoxytrityl (DMT) protecting group attached via the 5'-OH group of the nucleoside compounds.
  • the DMT group is shown as Structure I.
  • FIGS. 1 to 4 show the fluorescent excitation and emission spectra for selected representative phosphoramidite compounds which are commercially available. The chemical structures of these phosphoramidites are shown in Structures II to XI For these assays, commercial sources were used to obtain abasic phosphoramidite, ph-dC-Ac and ph-rC-Bz (Glen Research Co., Sterling, VA), and other phosphoramidites (PerSeptive Biosystems, Inc., Framingham, MA).
  • the excitation and emission fluorescent spectra for each of the compounds of Structures II to XI were determined in acetonitnle at two different concentrations Generally, a high concentration (0.07 M to 0.1 M) was chosen to be essentially equivalent to the concentration of the phosphoramidite that is pumped into a solid-support DNA synthesis column at the beginning of a coupling cycle. A low concentration (0.007 M to 0.01 M) was one-tenth the concentration of the high concentration and is essentially equivalent to the phosphoramidite concentration in a wash cycle that exists a solid-support DNA synthesis column at the end of a coupling cycle.
  • FIG. 1 shows the normalized excitation spectra after subtraction of background for phosphoramidites at 0.07 M in acetonitnle, over a range of 280 nm to 460 nm.
  • the phosphoramidites tested included abasic phosphoramidite and selected phosphoramidites having different protecting groups (tBDMSi, iBu, or Bz).
  • FIG. 2 shows the normalized emission spectra after subtraction of background for these phosphoramidites at 0.07 M in acetonitnle, over a range of 300 nm to 600 nm.
  • a nucleic acid base phosphoramidite such as ph-dT (Structure IV), which differs from the abasic phosphoramidite of Structure II only by the presence of the thymine base, exhibited characteristic excitation and emission spectra that differed from those of the abasic phosphoramidite (FIGS. 1 to 4).
  • the characteristic excitation and emission spectra of each nucleic acid base phosphoramidite probably results from an interaction of the base moiety with the DMT group, thereby shifting the fluorescence of the DMT group to a longer wavelength compared to the abasic phosphoramidite.
  • This compound differs from Structure IV only in the presence of the tBDMSi protecting group, which is not fluorescent, and the absence of the methyl group at the 5-pos ⁇ t ⁇ on carbon The absence of the hydrophobic methyl group would be expected to weaken the base's interaction with the DMT group, thus causing a smaller shift in the excitation and emission spectra compared to that of a thymine base. The smaller shift is shown in FIGS. 1 to 4.
  • phosphoramidites with characteristic excitation and emission spectra are two differently protected ph-dC compounds: ph-dC-Ac (Structure VI) and ph-dC-Bz (Structure VII) Both compounds have the same DMT-deoxy ⁇ bose structure, phosphate protective groups and a cytosme base, differing only in the protective group attached to the NH group (i.e., Ac in Structure VI and Bz in Structure VII). Neither of the NH-Ac or NH-Bz groups was fluorescent at the wavelength observed for these phosphoramidites. Both Structures VI and VII have excitation and emission spectra that differed from those of the abasic phosphoramidite and from each other.
  • excitation and emission spectra that distinguish between Structures VI and VII, as shown in FIGS. 1 to 4, probably result from interactions of the base and its respective protective group with the DMT moiety
  • excitation and emission spectra can be used to distinguish between compounds that vary only in a base-protective group.
  • each of the four phosphoramidites had a characteristic optimal excitation spectrum that differed from those of the other phosphoramidites.
  • Maximal excitation intensities were detected at about 310 to 320 nm for ph-dT, about 330 to 340 nm for ph-dG-iBu, about 360 to 370 nm for ph-dA-Bz, and about 370 to 380 nm for ph-dC-Bz.
  • the emission spectra for each of the phosphoramidites were determined by using a fixed excitation wavelength and measuring the fluorescence emission profile over a wavelength range.
  • Optimal emission wavelengths were detected at about: 305 to 315 nm for ph-dT, 325 to 335 nm for ph-dG-iBu, and 360 to 370 nm for ph-dA-Bz and ph-dC-Bz.
  • each phosphoramidite had characteristic excitation and emission spectra, featuring peak location, relative peak height, and line shape, which can be used to identify the phosphoramidite
  • the present method utilizes the distinguishable spectra of different phosphoramidites to detect and identify the compound used in individual coupling steps of nucleic acid syntheses.
  • the method includes determining the fluorescent excitation spectrum and/or emission spectrum of a sample solution containing at least one nucleic acid base phosphoramidite by measuring the relative intensity of fluorescence of the sample over a range of wavelengths.
  • the relative intensity of fluorescence may be measured using any of a variety of known devices that detect fluorescence in any of a variety of units (e g , counts per second (CPS), mV, or any other arbitrary unit for measuring fluorescence).
  • the excitation wavelength range has a lower limit of about 250 nm to 300 nm and an upper limit over the range of about 400 nm to 460 nm. More preferably the excitation spectrum is measured over a range of about 260 to 270 nm to about 400 to 440 nm, and most preferably the range is about 280 nm to about 460 nm.
  • the method alternatively or additionally includes determining the fluorescent emission spectrum following illumination at an excitation wavelength of a sample solution containing at least one nucleic acid base phosphoramidite by measuring the relative intensity of fluorescence of the sample at emission wavelengths over a range having a lower limit of about 250 nm to about 350 nm and an upper limit of about 550 nm to about 650 nm More preferably the emission spectrum is measured in a range of about 280 nm to about 600 nm, and most preferably the range is about 300 nm to about 550 nm For individual nucleic acid phosphoramidites, the fluorescent excitation
  • the optimal excitation spectrum for detecting that phosphoramidite in acetonitnle would be measured over a range of excitation wavelengths of about 260 nm to about 340 nm, although a range of about 275 nm to about 325 nm, or a narrower range of about 290 nm to about 310 nm is also suitable.
  • Routine testing of known compounds in known solvents using a selected detector system is used to generate a series of standards for determining peaks and optimal ranges for detecting and identifying the compound in the solvent used under experimental conditions.
  • the emission spectra of individual deoxynbonucleic acid phosphoramidites depend on the base (including the absence of a base in abasic compounds), the protecting group or groups and their positions on the phosphoramidite, and the solvent in the phosphoramidite is dissolved.
  • the characteristic emission spectrum of any known phosphoramidite compound in a known solvent can readily be determined by one skilled in the art routine testing procedures.
  • an emission spectrum is measured following excitation with a fixed excitation wavelength
  • the emission wavelengths preferably include a range of about ⁇ 40 nm surrounding a wavelength where a peak emission is expected for that phosphoramidite in the selected solvent at the selected excitation wavelength
  • the emission spectrum may be determined over a relatively narrow range (or at one or more individual wavelengths), such as for example, about ⁇ 25 nm surrounding the emission peak for the phosphoramidite compound to be detected.
  • An even narrower range for example, of about ⁇ 10 nm surrounding the emission peak for the phosphoramidite to be detected may be used.
  • Optimal excitation and emission spectra measurements for exemplary nbonucleic and deoxynbonucleic acid base phosphoramidites in acetonitnle are graphically shown in FIGS. 1 -4 Using the methods described herein, or using other well-known spectroscopy protocols, one skilled in the art may readily generate excitation and/or emission spectra for any known phosphoramidite compound in any solvent suitable for dissolving the selected phosphoramidite.
  • the time at which the fluorescent spectra are measured will depend on the sample measured (e.g., an influent solution, an effluent solution, a recylcmg influent solution, or a solution within a reaction chamber) and the flow rates of the chemicals within the synthesis system.
  • the fluorescent spectra (or an individual spectrum) are measured substantially simultaneous with dispensing an influent solution or recovering an effluent solution.
  • the fluorescence measurements are made within about 1 mm of dispensing an influent solution or recovering an effluent solution containing a phosphoramidite.
  • measurements are made within 0.25 mm to 0.5 mm of dispensing or recovering a solution.
  • Rapid measurements are particularly useful in an automated system of fluorescence measurement which is operationally attached to a nucleic acid synthesis system, to provide a real-time measurement of the individual coupling steps during a synthesis operation.
  • samples from a synthesis may be collected (e.g., one effluent sample per coupling step, collected in a suitable vessel, such as microtitre plate wells) and monitored later.
  • the scan time for measuring a fluorescent spectrum of a sample is generally dependent upon the capability of a detector device used for measuring. Preferably the scan time is less than 3 sec, more preferably is completed within about 1 sec, and most preferably is completed within mi seconds.
  • the measured fluorescent spectra of samples are compared to fluorescent spectra of known nucleic acid base phosphoramidites generated under the same or similar conditions (i.e., compared to standards)
  • the conditions include, but are not limited to, the solution components (e.g., solvent and other compounds or salts present in the solution) and their concentrations, temperature, time and duration of scan, and the excitation and/or emission wavelengths selected.
  • a practitioner of the present methods may rely on standards provided by others, or may determine the fluorescent spectra of the known phosphoramidites under controlled conditions that are standards for the practitioner's particular system.
  • a fluorescent spectrum is considered to include features such as peak location, peak intensity and line shape, which alone or cumulatively are characteristic of the nucleic acid base phosphoramidite present in the solution from which the fluorescent spectrum was generated.
  • Matching fluorescent spectra for identification does not require perfect correspondence between all features of an excitation or emission spectrum of a standard and the features of the effluent sample spectrum Generally, a correlation of one or more spectrum features (e g , peak location and/or line shape) is sufficient to distinguish one nucleic acid base phosphoramidite from another.
  • spectrum features e g , peak location and/or line shape
  • a method of detecting and identifying phosphoramidites in solution may involve generation of a fluorescent excitation spectrum, generation of a fluorescent emission spectrum, or detection of fluorescent emissions at discrete fluorescent excitation wavelengths.
  • a method of detecting and identifying phosphoramidites in solution may include generation of both a fluorescent excitation spectrum and a fluorescent emission spectrum for the same sample. Analysis of the combination of both spectra for a single sample is particularly useful for resolving mixtures of phosphoramidites in a single sample. Preferably, this combination method is performed by scanning substantially simultaneously the excitation and emission spectra of a single sample.
  • Substantially simultaneous scanning is preferred to ensure that the composition of the sample does not change during the scanning processes, for example, by dilution or mixing with another solution in an influent line, effluent line or a reaction chamber.
  • additional known photochemical properties of phosphoramidites in solution may also be used to detect and identify the compounds in a sample. These additional properties include, but are not limited to, fluorescent lifetimes, anisotropy or anisotropic decay. Qualitative or quantitative measurements of these additional photochemical properties may be particularly useful for resolving mixtures of phosphoramidites or distinguishing between phosphoramidites which have similar fluorescent excitation spectra and/or fluorescent emission spectra.
  • this method of fluorescence spectroscopy identification of nucleic acid phosphoramidites is performed with a detection system that is attached directly or indirectly to a nucleic acid synthesizer.
  • directly joining a fluorescence detector to a synthesizer is meant that no intermediate device separates the devices in the system
  • the detector may be joined to an influent line or an effluent line of a synthesizer, or is positioned to directly detect solution within a fluorescent-transparent reaction chamber.
  • the fluorescence detector may be indirectly joined to the synthesizer by one or more linking components, such as a diverter line or pump that moves a sample of influent solution (single or recycling), effluent wash or reaction components to another location for fluorescence detection
  • the detection system is automated so that measurement of fluorescent spectra is triggered by the synthesizer, for example, by electronic detection of sample movement, or at a predetermined time after a coupling reaction is initiated, or when an effluent wash is delivered to an effluent line.
  • the operator may manually compare the measured fluorescence to known standards (e.g., fluorescent spectra of known phosphoramidites) to the determine the presence and identity of the phosphoramidite in the sample.
  • known standards e.g., fluorescent spectra of known phosphoramidites
  • the comparison and/or determining steps may be automated, such as by using computer operations defined by one or more algorithms that can readily be determined by those skilled in the art. For example, automated comparison and/or determining steps similar to those used in automated DNA sequencing devices may be employed.
  • An automated system for measuring fluorescent spectra or individual measurements, comparing fluorescent spectra or individual measurements with known spectra or individual measurement and determining the identity and/or quantity of a phosphoramidite in a solution associated with a nucleic acid synthesis coupling step is particularly useful for monitoring the coupling yields for each coupling reaction in a nucleic acid synthesis.
  • the system determines both quantity and identity of the phosphoramidite in the sample. For example, by using these methods and automatically detecting the amount (e.g., based on the fluorescent intensity detected) and identity of a phosphoramidite present in an effluent sample, the relative coupling efficiencies of each of the individual coupling reactions are determined.
  • the quality of the final synthetic nucleic acid product is predicted using the methods of the present invention, thereby eliminating the need for a separate analysis step. That is, little or no post-synthetic analysis of the product itself would be required to determine the quality of the product. Moreover, if a problem is detected during a synthesis (such as an inefficient or ineffective coupling step), the synthesis may be modified immediately or terminated before more reagent is consumed.
  • EXAMPLE 1 Fluorescence Measurements at Fixed Points to Identify Phosphoramidite Compounds This example shows that fluorescence measurements made at a series of excitation wavelengths, rather than measurements over an entire spectrum of excitation wavelengths, may be used to identify a phosphoramidite compound in a solution.
  • the fluorescence emission in CPS was determined for each of the four deoxynbonucleic acid base phosphoramidites of Structure IV ("ph- dT”), Structure XI (“ph-dG-iBu”), Structure X (“ph-dA-Bz”) and Structure VII (“ph-dC-Bz”) at different fixed excitation wavelengths of 313 nm, 333 nm, 365 nm and 377 nm.
  • excitation at 313 nm, 333 nm, 365 nm and 377 nm of an acetonitnle solution containing ph-dT resulted in maximal fluorescent emission at excitation wavelength 313 nm
  • excitation under the same conditions of an acetonitnle solution containing ph-dG-iBu resulted in maximal fluorescent emission at an excitation wavelength of 333 nm, thus distinguishing between these two compounds.
  • a Jasco FP-920 fluorescence detector was attached via a Dionex UI20 Universal Interface to the DMT effluent line of a Milligen/Biosearch Model 8750 synthesizer, and the post-coupling washes from the synthesizer were redirected to this line.
  • the synthesizer was configured to send an electrical signal to initiate or trigger data acquisition on the fluorescence detector unit
  • FIG. 5 shows a typical profile of the fluorescence signal obtained from a single post-coupling wash (i.e., effluent sample) for the deoxynbonucleic acid phosphoramidite ph-dC-Bz, which was present at a concentration of 0.07 M in the influent solution used for the coupling step.
  • a single post-coupling wash i.e., effluent sample
  • an abbreviated wash protocol was used consisting of three washes of 1.5 sec each, instead of three washes of 30 sec each that are generally used with this system.
  • the excitation wavelength was 385 nm and the emission wavelength was 460 nm.
  • a typical spectrum measurement at a rate of 100 nm/sec allows an excitation scan (e.g., 300 nm to 400 nm) to be completed in one second.
  • excitation scan e.g., 300 nm to 400 nm
  • fluorescence detector systems e.g., Jasco FP-920 detector
  • the methods of the present invention can be practiced by those skilled in the art using well known technology and devices.
  • FIG. 6 shows the results of fluorescent excitation spectra that were obtained during the post- coupling washes obtained from an oligonucleotide synthesis reaction performed substantially as described above for FIG. 5
  • FIG. 6 presents on a single graph the multiple spectra obtained from multiple wash samples, each representing a single phosphoramidite compound.
  • the emission wavelength was fixed at 420 nm
  • the excitation wavelengths were over a range of 275 nm to 400 nm
  • the scan time was 3 sec.
  • the four phosphoramidites, ph-dA-Bz, ph-dT, ph-dG-iBu and ph- dC-Bz were distinguishable from one another, and from pure acetonitnle ("MeCN") based on the spectra shown in FIG. 6. That is, based on the peak positions, peak intensities, and shapes of the lines defining the different excitation spectra, the individual phosphoramidites were distinguished in effluent obtained from an oligonucleotide synthesis reaction device (Milligen/Biosearch Model 8750). The spectra obtained using the effluent correlated with the results of the excitation spectra shown in FIGS. 1 and 3. Referring to FIG.
  • the maximal intensity for ph-dT (— 4 — ) was between 300 nm and 325 nm, peaking at about 310 nm.
  • the maximal intensity for ph-dG-iBu (— ⁇ — ) was observed between 310 nm and 345 nm, with the peak at about 325 nm.
  • the maximal intensity was a relatively broad peak between 320 nm to 360 nm, peaking at about 350 nm
  • the dT phosphoramidite showed the most intense fluorescence under these conditions.
  • the fluorescent signal obtained for the acetonitnle spectrum may have resulted from the presence of an activator solution (0.45 M 1 H-tetrazole in anhydrous acetonitnle; Glen Research, Sterling, VA) that is mixed with the phosphoramidite monomers during the coupling reactions.
  • FIG 7 shows the effect of time on the excitation spectra obtained for one phosphoramidite, ph- dC-Bz.
  • the excitation spectra for ph-dC-Bz in synthesizer effluent were measured at different times (0.15, 0.2, 0.25, 0 3, 0.35 and 0 4 m after the detector was triggered to begin measurement by the electronic signal from the synthesizer).
  • the spectra resembled the acetonitnle spectrum (e.g., see FIG.
  • the spectra exhibited a peak at about 332 nm. As shown in FIG. 7, the spectra obtained at 0.25 and 0.3 mm most closely resembled the spectra obtained for ph-dC-Bz as shown in FIGS.
  • FIG. 8A to 8H show the individual excitation spectra obtained for sequential effluent obtained during synthesis of the oligonucleotide having the sequence 5' AACGGTTCT 3', starting from base 2, adjacent to the 3' T residue attached to the solid support, and continuing to base 9, the 5' residue of the oligonucleotide. That is, FIG. 8A shows the spectrum for effluent following base 2 addition (i.e., immediately adjacent to the 3' base), FIG. 8B shows the spectrum for effluent following base 3 addition, FIG. 8C shows the spectrum for effluent following base 4 addition, FIG. 8D shows the spectrum for effluent following base 5 addition, FIG.
  • FIG. 8E shows the spectrum for effluent following base 6 addition
  • FIG 8F shows the spectrum for effluent following base 7 addition
  • FIG. 8G shows the spectrum for effluent following base 8 addition
  • FIG 8H shows the spectrum for effluent following base 9 addition, the 5' base of the oligonucleotide.
  • the resulting spectra were compared to reference spectra for each of the four phosphoramidites (e.g., see FIGS 1 , 3 and 6) to determine the identity of the excess phosporamidite present in the effluent for each synthetic coupling step.
  • the spectrum shown in FIG. 8A obtained for the effluent of the first coupling reaction did not provide meaningful results, probably due to gas present in the line conducting the effluent to the detector.
  • FIGS. 8B and 8C show bases 3 and 4, respectively, were T additions
  • FIGS. 8D and 8E show bases 5 and 6, respectively, were G additions
  • FIG. 8F shows base 7 was a C addition
  • FIGS. 8G and 8H show bases 8 and 9, respectively, were A additions.

Abstract

Procédés de détection et d'identification de phosphoramidites à une base d'acide nucléique individuelles dans une solution échantillon par appariement de spectres d'excitation et/ou d'émission de fluorescence avec des spectres similaires de phosphoramidites connues. Des procédés permettant de déterminer la précision de la synthèse d'acide nucléique par analyse des spectres de fluorescence d'échantillons (influent, effluent, mélanges de réaction) associés à des étapes de liaison individuelles qui utilisent les phosphoramidites à une base d'acide nucléique, et d'identifier la phosphoramidite contenue dans chaque échantillon sont également décrits.
PCT/US2000/023422 1999-08-27 2000-08-25 Procede d'identification de phosphoramidites a une base d'acide nucleique par la spectroscopie a fluorescence WO2001016371A2 (fr)

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WO1995026416A1 (fr) * 1994-03-25 1995-10-05 Research Corporation Technologies, Inc. Procede de diagnostic par biocapteurs d'acide nucleique
WO1997036960A1 (fr) * 1996-04-01 1997-10-09 The Perkin-Elmer Corporation Colorants asymetriques a base de benzoxanthene
WO1999013110A1 (fr) * 1997-09-11 1999-03-18 Seq, Ltd. Procede de fabrication de photoproduits de nucleotides fluorescents pour sequençage d'adn et analyse
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WO1994001214A1 (fr) * 1992-07-06 1994-01-20 Beckman Instruments, Inc. Procede de surveillance de la reaction et de l'ecoulement d'un processus en ligne
WO1995026416A1 (fr) * 1994-03-25 1995-10-05 Research Corporation Technologies, Inc. Procede de diagnostic par biocapteurs d'acide nucleique
WO1997036960A1 (fr) * 1996-04-01 1997-10-09 The Perkin-Elmer Corporation Colorants asymetriques a base de benzoxanthene
WO1999013110A1 (fr) * 1997-09-11 1999-03-18 Seq, Ltd. Procede de fabrication de photoproduits de nucleotides fluorescents pour sequençage d'adn et analyse
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