Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6848—Methods of protein analysis involving mass spectrometry
• • 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
  • C12Q1/6872—Methods for sequencing involving mass spectrometry
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
  • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
  • Y10T436/143333—Saccharide [e.g., DNA, etc.]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

• This invention pertains to the post-synthetic analysis and validation of oligonucleotides, specifically the post-synthetic analysis and validation of a plurality of oligonucleotides.
• Oligonucleotides are used in a multitude of research and clinical applications. Ensuring the integrity of the oligonucleotide is vital to prevent failures or misleading results in applications using the oligonucleotides. Several methods have been developed to ensure that the resulting oligonucleotide is what the technician intended for use in their application. [0004] One method of ensuring quality during the synthesis of the oligonucleotide is through trityl monitoring.
• the dimethoxytrityl (DMT) group that is used for capping the 5'- hydroxyl group of the monomers in the oligonucleotide synthesis fluoresces in its protonated form after it is removed with an acid. The absorbance of the fluorescence can be measured at or around 498 nm. A decrease in the absorbance level can be an indication that coupling was inefficient.
• oligonucleotide target can be assessed post-synthetically by measuring the predominant molecular weight of the population.
• the target sequence is known, so the calculated molecular weight of the bases themselves is the standard by which one compares the measured molecular weight to see if the desired compound was created.
• One way of utilizing this principle is through matrix assisted laser desorption ionization time-of- flight mass spectrometry (MALDI-TOF).
• MALDI-TOF uses laser light in conjunction with a chemical matrix to impart a charge to the sample in question and repel it from the sample plate. The resulting ions travel through a flight tube to the detector, which measures particle counts as a function of time.
• the time-of- flight (TOF) is directly proportional to the mass of the molecule.
• MALDI-TOF is a robust and incredibly high-throughput process for assessing molecular weight.
• One drawback of MALDI-TOF is that the ionization efficiency (and therefore the resolution) of the procedure drops rapidly above ⁇ 45 bases or > 13, 000 Da.
• the popularity of 70 mer arrays and long oligonucleotides for cloning and/or gene synthesis another method is needed to assess longer products.
• Electrospray ionization (ESI) mass spectroscopy ionizes target molecules such as oligonucleotides into multiple charge states. The readout of these charge states is a waveform that can be deconvoluted into parent peaks. The method uses a tight m/z window of 500 — 1,500, which gives it high mass accuracy. As only the charge state will vary for the ions, oligonucleotides with high molecular weights can be analyzed using this method. Therefore ESI is often a preferred quality control (QC) method over MALDI-TOF for longer oligonucleotides (see Elliott, B. and Hail, M. High-Throughput Analysis of Oligonucleotides Using Automated Electrospray Ionization Mass Spectrometry American Biotechnology Laboratory, January 2004).
• QC quality control
• the proposed method involves generating a theoretical MALDI-TOF or ESI trace (fingerprint) of a multi-oligonucleotide sample and then comparing the actual MALDI-TOF or ESI data of the mix to the fingerprint as a QC check.
• the proposed method involves generating a theoretical ESI trace (fingerprint) and then comparing the actual ESI trace data of the mix to the fingerprint to verify the accuracy of the syntheses.
• the method enables multiple data points from the ESI data to be graphically represented even if more than one oligonucleotide sequence in the assay shares the same data point.
• the theoretical trace assigns a signal value of one to each discrete mass, i.e. a molecular weight range that has no overlap with another range would be assigned a value of one. If more than one mass appears at a respective molecular weight, the intensity is increased by that number (e.g.
• the range would be assigned an intensity value of 3).
• the number of peaks would be further pared back by assuming that any that crossed at 50% or more of the peak height would be additive.
• the width of the peak is dependent on the length of the sample oligonucleotide.
• the new peak's mass would be calculated using the following formula:
• MWnew (MWi intensity i + MW 2 *Intensity 2 + ...MW n *Intensity n ) / (Intensityi +
• the new peak intensity is simply the addition of the individual intensities.
• the inventive method is not limited to any particular assay format, and it could also be used to fingerprint MALDI-TOF results.
• Figure 1 is a theoretical trace generated from the data in Table 1 of Example 1 using Microsoft Excel ® .
• Figure 2 is an actual ESI trace of the 48 samples in Example 1.
• Figure 3 is a theoretical trace generated from the data in Table 1 of Example 1 using Data ExplorerTM software from Applied Biosystems.
• Figure 4 is an overlay of the theoretical trace in Figure 3 and the actual ESI data from Figure 2.
• Figure 5 is a graph of the data in Table 1 wherein the intensity is equal to the amount of oligonucleotide samples with the same molecular weight.
• Figure 6 is a magnified version of the data outlined in Figure 5.
• Figure 7 depicts the amount of overlap between peaks (2 + 3) and 4 to illustrate why peak 4 is combined with peaks 2 and 3 to form one peak in the theoretical trace in Figure 1.
• Figure 8 illustrates the gap (no overlap at greater than 50% peak height) between peaks (5 + 6) and (7 + 8) that would require separating the two peaks for the theoretical trace.
• the proposed method involves generating a theoretical ESI trace (Fingerprint) and then comparing the actual ESI data of the mix to the fingerprint as a QC check.
• the method enables multiple data points from the ESI data to be graphically represented even if more than one oligonucleotide sequence in the assay shares the same data point.
• the theoretical trace assigns a signal value of one to each discrete mass, i.e. a molecular weight range that has no overlap with another range would be assigned a value of one. If more than one mass appears at the respective molecular weight, the intensity increases by that number (e.g.
• MW new (MWi intensity i + MW 2 *Intensity 2 + ...MW n *Intensity n ) / (Intensity i + Intensity 2 + ... Intensity n )
• a sample containing each of the oligonucleotides of interest is prepared (a "multi-oligonucleotide sample"), and the resulting sample is run through the ESI instrument. Peaks representing the oligonucleotides in the sample would be present, and there would be higher peaks at points where multiple oligonucleotides have the same or nearly the same molecular weights.
• a standard fingerprint could represent a standard assay kit wherein the oligonucleotide set is always the same.
• the invention can be incorporated into a processor to provide for an automated verification of the oligonucleotide mixture.
• the algorithm in Formula 1 can be a component of software that would process the given molecular weights of the individual oligonucleotide constituents that are expected to comprise an oligonucleotide mixture into the calculated peak or set of peaks.
• the processor can work with an instrument that provides a measured mass spectrum to provide an automated system to determine whether the measured mass spectrum aligns with the calculated spectrum.
• multiple oligonucleotides refers to more than one oligonucleotide sample, wherein the samples may or may not have the same molecular weight, and the samples may or may not have the same sequence.
• Example 1 contains 48 oligonucleotide samples, i.e. a multiple oligonucleotide sample with 48 oligonucleotides.
• the terms “mass spectrometry”, “mass spectrum” and “mass assessment” encompass a number of technologies for both ionization methods and mass analysis.
• mass spectrometry formats include MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time of Flight); ESI-TOF (Electrospray Ionization Time of Flight); ESI (Electrospray Ionization generally with a single or triple quadrupole(s)); ESI-QIT (Electrospray Ionization Quadrupole Ion Trap (Linear or 3-D)); MALDI-QIT (Matrix Assisted Laser Desorption Ionization Quadrupole Ion Trap (Linear or 3-D)); MADLI- QTOF (Matrix Assisted Laser Desorption Ionization Quadrupole Time of Flight); and ESI- QTOF (Electrospray Ionization Quadrupole Time of Flight).
• MALDI oligonucleotide
• ESI Quadrupole Ion Trap
• This example demonstrates the accuracy of the theoretical fingerprint method using a 48-oligonucleotide sample.
• the expected molecular weight of each oligonucleotide was calculated and entered into Table 1. As shown in Table 1, 19 oligonucleotides have the same molecular weight. The peaks were calculated to be 20 Da wide to coincide with the general width of the sample oligonucleotides, and overlapping peaks were combined.
• Figure 1 shows a chart of the data from Table 1 using Formula 1 using Microsoft ® Excel ® .
• the first step in designing the theoretical trace was to first combine the oligonucleotide samples that had the same molecular weights by assigning intensities equal to the number of samples that had the same molecular weight. Then Formula 1 was applied to all peak sets until the overlap was less than 50% between all peaks To illustrate how the peaks were further combined to result in the Figure 1 trace, a subset (outlined in Figure 5) of the data was chosen to explain the next step.
• Figure 6 is a magnified version of the data outlined in Figure 5. Peaks 1 and 11 did not overlap any other peaks and therefore they remained independent peaks. The peaks where there was overlap at greater than 50% peak height were combined using Formula 1. For example, peaks 2, 3 and 4 would be combined because they all overlap at greater than 50% peak height.
• Peaks 5 and 6 were combined to form 1 peak, and peaks 7 and 8 were combined to form another peak. Although peaks 6 and 7 overlap at greater than 50% peak height, the respective sets of peaks (5 + 6) and (7 + 8) had a greater overlap than 6 and 7 do to each other. Once the greater overlap peaks were calculated ((5 + 6) and (7 + 8)), there was no overlap greater than 50% between (5 + 6) and (7 + 8) and therefore were not combined. See Figure 8.
• FIG. 1 Another theoretical chart was generated using Formula 1 with the data from Table 1 with DataExplorerTM software from Applied Biosystems ( Figure 3).
• Figure 3 A sample containing all 48 oligonucleotides was loaded into an LCQTM ESI instrument from Thermo Fisher Scientific to generate the actual trace.
• Figure 2 contains the actual ESI trace data.
• Figures 2 and 3 were overlaid ( Figure 4) to demonstrate the correlation between the expected and actual peak values. The results demonstrate that each peak in the actual trace has a corresponding peak in the theoretical traces, and the actual trace contains each peak that was present in the theoretical fingerprints.

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• 2007
  • 2007-10-23 JP JP2009534809A patent/JP2010508024A/ja active Pending
  • 2007-10-23 US US11/876,960 patent/US7604998B2/en active Active
  • 2007-10-23 CA CA002667374A patent/CA2667374A1/en not_active Abandoned
  • 2007-10-23 WO PCT/US2007/082197 patent/WO2008070314A2/en not_active Ceased
  • 2007-10-23 AU AU2007329729A patent/AU2007329729A1/en not_active Abandoned
  • 2007-10-23 EP EP07871211A patent/EP2089706A4/en not_active Withdrawn
• 2009
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