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WO1989002475A1 - Polynucleotides polyaldehydiques utilises comme sondes, leur preparation et utilisation - Google Patents

Polynucleotides polyaldehydiques utilises comme sondes, leur preparation et utilisation Download PDF

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Publication number
WO1989002475A1
WO1989002475A1 PCT/GB1988/000754 GB8800754W WO8902475A1 WO 1989002475 A1 WO1989002475 A1 WO 1989002475A1 GB 8800754 W GB8800754 W GB 8800754W WO 8902475 A1 WO8902475 A1 WO 8902475A1
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polynucleotide
dna
probe
hybridization
groups
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PCT/GB1988/000754
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English (en)
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Thomas Lee Mattson
Robin Ewart Offord
Keith Rose
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Thomas Lee Mattson
Robin Ewart Offord
Keith Rose
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Publication of WO1989002475A1 publication Critical patent/WO1989002475A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • This invention relates to polyaldehydic polynucleotides in use as probes, their preparation and use.
  • the invention is particularly concerned with, but not limited to, polynucleotides having reporter groups attached thereto.
  • literature references are indicated by numbers in parenthesis.
  • RNA and DNA molecules may be useful in a number of practical applications. Many of the currently envisaged applications involve hjybridization of these modified nucleic acid molecules (hereafter called “probes”) to complementary DNA and RNA sequences (hereafter called “targets”).
  • probes modified nucleic acid molecules
  • targets complementary DNA and RNA sequences
  • This class of potential applications includes DNA-based diagnostics, use as anti-messages to modulate levels of gene expression (1), and as a component of novel methods for cleaving DNA or RNA chains at any specific site (2).
  • the probes are modified prior to hybridization with the target.
  • reporter groups entities added to the DNA probe have been called reporter groups. They function, inter alia, as "labels" to permit the probes to be traced.
  • reporter groups are preferred (3).
  • reporter does not adequately describe all the functions envisaged for the entities added to the nucleic acid molecules, this term is used loosely herein to refer to any entity artificially attached, covalently or not, to the probe molecules.
  • the probe molecules are labelled with the reporter groups prior to hybridization with the target sequences. This is done because it is usually difficult selectively to label only the probe component of the probe-target hybrid molecules after hybridization (some exceptions are discussed below).
  • reporter groups attached to nucleic acid probes prior to hybridization with the target sequences should fulfill the following five criteria:
  • the reporter group should not interfere with the hybridization process to any serious extent.
  • the reporter group should not decrease the thermal stability of the probe-target hybrid molecules.
  • the reporter group must not induce non-specific binding of the probe to the target sequences.
  • biotinylated probes Some of the limitations of the currently available methodologies can be illustrated with biotinylated probes. In these probes the biotin is present prior to hybridization. However, in practice one never achieves the theoretically maximum sensitivity, which would be to attach biotin to all of the biotin-accepting bases in the probe molecule. That is, in currently available methods, the biotin is not attached to all of the biotin-accepting bases (37, 38). Thus, biotin as a reporter group illustrates a general problem associated with currently available probes: how can one attach reporter groups to all of the biotin (or reporter)-accepting bases in a probe molecule, particularly post hybridization.
  • the present invention solves the problem of how to attach reporter groups to all of the reporter-accepting bases and thus solves the general problem of how to achieve the theoretical maximum sensitivity. Consequently, with the present invention, one can attach biotin to all of the biotin-accepting bases in a way that avoids the problems encountered when biotin is added to all of the biotin-accepting bases prior to hybridization.
  • European Patent Specification No. 0133473 is concerned with in vivo labelling of polynucleotide probes and in Example II thereof describes, in isolation, the biotinylation of an unidentified "T4 DNA".
  • the method described uses periodate oxidation, the method is not correlated in any way with hybridization work nor with a particular probe or probes. Neither is described.
  • Hybridization data is critical to use of any material in or as a polynucleotide probe, and there is no such data present. It is not even possible to be certain of the nature of the "T4 DNA" used and there is no disclosure whatsoever of its use.
  • nucleic acid molecules contain more than a single pair of chemically reactive aldehyde groups and these, in contrast to the present invention, are derived from a sugar component of the nucleic acid and not from non-backbone residues.
  • RNA molecules in which the ribose moiety at the 3' end of the polynucleotide backbone has been oxidized (5). They will be discussed further below.
  • Numerous kinds of compounds can be covalently attached by chemical methods to the aldehydes of the polyaldehydic products produced according to this invention.
  • the invention enables conversion to aldehydic form before hybridization (followed by labelling with reporter groups).
  • the advantages of this technique are considerable.
  • the end user of the probe does not have to employ an oxidation step; a pre-oxidized probe can be used.
  • RNA sequences may be detected by post-hybridization labelling techniques that employ covalent bonds. Not only is labelling prior to hybridization avoided, but chemical attack, of the RNA is also avoided since a pre-oxidized probe is used. This is an important consideration because oxidation and labelling after hybridization with a population of RNA. molecules would usually result in the oxidation of the ribose moiety at the 3' ends of RNA chains and the production of a single pair of aldehydes.
  • RNA molecules that are not “targets”, and which are in vast excess over the true “targets”, would thus also become labelled with the aldehyde reactive reporter groups, there would be an increased level of "noise” resulting from. this non-specific labelling.
  • the target molecules are DNA
  • the reporter molecule can be attached to the probe at a much lower temperature than was used for the hybridization, reporter-probe-target complexes can be produced which will be stable at, say, room temperature, but which would have been very unstable had they been formed at th ⁇ elevated temperatures required for hybrid formation.
  • One consequence of this approach is that there may be no limit on the number of reporter molecules that can be attached to each probe molecule.
  • This invention unexpectedly provides new opportunities for increasing the sensitivity of detection employing previously used DNA detection systems, new methods, compounds and chemistry for linking reporter groups to DNA probes and new opportunities for developing entirely novel detection systems for diagnostics.
  • Detection systems, or components of detection systems exploited previously only in protein-based diagnostics can be used.
  • one of the antigenic components of such systems, such as insulin could be directly attached to the polyaldehydic probes after hybridization.
  • the ability to produce pairs of aldehydes very close to one another on a DNA chain should make it possible to significantly increase the signal in systems involving two components that must physically be closely associated, such as energy transfer systems (34).
  • a related idea should permit a significant enhancement, not of signal strength itself, but of the signal-to-noise ratio.
  • a characteristic feature of probes labelled according to this invention is that, distributed along the chain according to the nucleotide sequence, some labels will when seen in three dimensions, be in pairs of clusters.
  • polymers previously unused in DNA-based diagnostics containing, for example, but not limited to, amino acids (e.g., derivatives of polylysine or polyglutamic acid) or sugar moieties (e.g., polysaccharides or even another polyaldehydic DNA molecule) or amino-alcohols.
  • polymers e.g., derivatives of polylysine or polyglutamic acid
  • sugar moieties e.g., polysaccharides or even another polyaldehydic DNA molecule
  • amino-alcohols e.g., amino
  • O-alkyl-hydroxylamine reagents are particularly attractive because they are highly specific for aldehydes, because they form adducts rapidly and because, unlike reagents containing aliphatic or aromatic amines, they form stable adducts with polyaldehydic DNA without the use of additional reagents, e.g., cyanoborohydride, to reduce the Schiff-base intermediate between aldehydes and amines.
  • additional reagents e.g., cyanoborohydride
  • Polyaldehydic probes also provide the opportunity to create entirely new detection and amplification systems. For example, one could attach compounds which can act as nucleation centers for the stable polymerization of protein or other kinds of monomers into large structures.
  • One example of such a compound is Phalloidin and chemically related compounds, which can nucleate the polymerization of actin monomors into long filaments (F-actin) (35). This would create a bundle of actin filaments around each probe-target hybrid molecule. Since actin bundles can be seen in the light microscope, one might be able, using either native or fluorescent labelled actin monomers, to quantitatively determine the absolute number of target molecules in the sample being analyzed.
  • polyaldehydic probes can provide a number of surprising advantages that were previously unrealized with any kind of DNA probe.
  • Another surprising advantage of the methods of this invention is that the aldehydes can be selectively produced, favoring those probe molecules that have hybridized with the target sequences.
  • this invention can both simplify the procedure used in the diagnostic process and provide a new and novel solution to the problem of how to reduce the noise (the non-specific signal) coming from the excess, unhybridized probe molecules still present after the hybridization step.
  • this separation step could be eliminated entirely from the diagnostic procedure, or as a minimum, one would have a reduced level of "noise” after the separation step.
  • This can be done according to the present invention by hybridizing with probe molecules that contain the glucose-accepting base, 5-hydroxymethylcytosine (HMC), but which are not linked to either glucose or to the aldehyde pairs produced by oxidation of the glucose.
  • the glucosyl transferase enzymes which transfer glucose from UDP-glucose to the glucose-accepting bases, preferentially add glucose to HMC bases that are in double stranded DNA over HMC bases that are in single stranded DNA (17, 18).
  • the aldehydes will be produced preferentially on those probe molecules that have hybridized with the targe sequences. Since the aldehyde-reactive reporter groups will not efficiently attach to the unhybridized probe molecules, one could just ignore the unhybridized probe molecules and not separate them from the probe-target hybrids. Alternatively, one could still perform the separation step and have a reduced level of "noise".
  • this method can simplify the procedure, reduce the noise level and solve the problem of how to remove the unhybridized probe molecules.
  • polyaldehydic DNA also offers an entirely different and previously unexplored chemical basis for attaching reporter or other molecules to DNA probes prior to hybridization with target sequences. Since the physical and chemical properties of polyaldehydic DNA are different from any previously employed DNA probes, molecules previously considered unsuitable as reporter molecules with other types of probes might well be usable with polyaldehydic probes, e.g. molecules which are incompatible with the use of DNA polymerases can be employed and molecules which are incompatible with the conventional use of blocking groups in nucleotide synthesis. In general, reporter groups can now be used which cannot be used prehybridization. The different chemical options provided by the presence of aldehyde groups opens up new possibilities.
  • RNA-DNA hybrids are immunologically distinct from either RNA-RNA or DNA-DNA duplexes and thus can also be specifically labelled after the hybridization step (7).
  • the reporter molecules would not be added to the hybrid per se, but rather only to the aldehyde groups present uniquely on the probe strand of the probe-target hybrids and they can be added chemically, not immunologically .
  • the present invention applied to DNA-diagnostics is also distinct from situations where bases on the probe molecules can be chemically modified prior to hybridization to make them antigenic (8,9). In these situations, entities can be attached directly to the probe strand of the probe-target hybrids, but only by immunological methods.
  • polyaldehydic polynucleotides prepared according to the principles and methods of this invention are not limited to DNA-based diagnostics and other situations involving nucleic acid hybridizations.
  • the polyaldehydic polynucleotide is potentially useful because it can perform two functions: it can be part of a duplex molecule with complementary nucleic acid sequences and it can serve as a physical structural support for the attachment of reporter molecules.
  • the important quality of the polyaldehydic products may primarily be their ability to function ass as physical structure or support for the attachment of various entities or for linking various entities.
  • polyaldehydic DNA can be used as a support for immobilizing enzymes or as a protein cross linking agent. Wool cross linked with bifunctional aldehyde molecules is reported to have altered properties (10). Wool fibres cross linked by polyaldehydic DNA may have other novel and useful properties. Polyaldehydic DNA can also serve as a polyvalent aldehydic linker for the attachment of numerous toxin or other useful entities to monoclonal antibodies (6), opening up the prospect of many new drugs and therapeutics by employment of techniques in principle well known in the field of immunotoxins or other "target specific" drugs.
  • the invention provides a polynucleotide probe having a base sequence hybridizable to a preselected polynucleotide and also having a multiplicity of reporter groups each linked thereto via an aldehyde group which has been provided by oxidation of a non-backbone sugar residue.
  • the invention also includes a duplex comprising a polynucleotide hybridized to a polynucleotide probe designed to detect a chosen base sequence or polynucleotide, the probe carrying a multiplicity of aldehyde groups or a multiplicity of reporter groups each linked to the probe by an aldehyde group, and, in either case, the aldehyde groups each being provided by oxidation of a non-backbone sugar residue.
  • a further aspect of the invention is a duplex comprising a polynucleotide hybridized to a polynucleotide probe designed to detect a chosen base sequence or polynucleotide, the probe carrying intact non-backbone sugar residues so as to provide means for identifying the duplex by oxidizing said sugar residues to provide aldehyde groups to which reporter groups may be attached.
  • a process for preparing a polynucleotide probe which comprises providing a polynucleotide having a base sequence hybridizable to a preselected polynucleotide and also in which there are non-backbone sugar residues and oxidising the said residues to provide aldehyde groups.
  • the invention includes a non-T4 derived polyaldehydic polynucleotide having aldehyde groups provided by at least one oxidized non-backbone sugar residue.
  • Such polynucleotides can be used, e.g. as a support for an immobilized enzyme, as a protein cross-linking agent, or as a aldehydic linker, e.g. wherein the polynucleotide is employed to link a toxin to a monoclonal antibody or to link a molecule having a biological effect on target cells to a monoclonal antibody for those cells.
  • the invention provides a method of diagnosis not being one practised on the human or animal body and in which a probe as defined above is employed to detect by hybridization a polynucleotide characteristic of a disease or disorder or stage thereof.
  • the invention also includes the following methods:
  • the nucleic acid used for specifically demonstrating the principles disclosed herein is biologically produced glucosylated DNA.
  • the scope of this invention is not limited to this kind of nucleic acid molecule.
  • Suitable DNA (or RNA) molecules can be produced entirely or partially in vitro. Since these synthetically produced nucleic acids can be treated according to the principles disclosed here, they are included in the scope of this invention. We will briefly describe some approaches that can be employed to produce suitable DNA or RNA molecules in vitro.
  • glucosylated DNA of the T-even family of bacteriophages is produced as the end result of a complicated enzymatic process that can be summarized as involving two distinct steps: (1) incorporation of a glucose-accepting nucleotide into a polynucleotide chain followed by (2) the covalent addition of one or two glucose molecules to the glucose-accepting nucleotide.
  • the critical consideration for this invention is that either of these steps can be performed in vitro.
  • the glucose accepting nucleotide contains the unusual base 5'-hydroxymethylcytosine (HMC).
  • HMC replaces all of the cytosines that are normally present in most DNA species.
  • glucose can be attached to the 5' -hydroxymethyl group of these HMC bases through the action of enzymes that are called glucosyl transferases.
  • cytosine and hydroxymethylcytosine in the same polynucleotide chain. If it is important precisely to control the position(s) of the glucose-accepting HMC bases along the nucleic acid chain, synthetically produced polynucleotide chains might be preferable to those produced biologically. That is, only with in vitro methods would it always be possible precisely to control where the glucose accepting and non-accepting cytosines would be located. Glucose can then be added, in vitro, to these synthetically produced DNA molecules.
  • this invention relates to polynucleotides containing one or more, oxidizable, non-backbone, sugar moieties.
  • RNA chains containing the unusual base In vivo, glucose is attached to DNA chains containing the unusual base, hydroxymethylcytosine (HMC).
  • HMC hydroxymethylcytosine
  • RNA chains containing modified nucleotides can be produced enzymatically and chemically (13).
  • RNA polymeases or synthetases might also be produced in vitro with some RNA polymeases or synthetases. That is, some RNA polymerizing enzyme might incorporate ribonucleotides bearing the unusual base HMC into polynucleotide chains. Glucose might then be added in vitro to these synthetically produced RNA chains by a glucosyl transferase enzyme.
  • RNA chains containing the glucose-accepting base, HMC have not been produced, one can reasonably anticipate that a glucosyl transferase enzyme, or a mutant varient of such an enzyme, will be able to transfer glucose, under standard or altered reaction conditions, to 5'hydroxymethyl-cytosine containing RNA chains that are in a single stranded state or in a hybrid structure with either another RNA chain or in a hybrid with a DNA chain. That is, the activity of these enzymes, which requires a modified base, may be relatively indifferent to whether the backbone of the polynucleotide chains are composed of ribose or deoxyribose.
  • the glucose accepting base, HMC is a pyrimidine carrying a hydroxymethyl group at the 5' position of the pyrimidine ring. Recognition of this 5' hydroxy methyl group must therefore be important for the activity of the glucosyl transferase enzymes.
  • the presence of a 5' hydroxymethyl group on a pyrimidine ring may be the only or the major structural requirement for the activity of one or more of these transferase enzymes.
  • 5' hydroxymethyl-pyrimidine bases for example, 5' hydroxymethyluracil, a natural constituent of the DNA of a number of Bacillis subtilis phages (15)
  • 5' hydroxymethyluracil a natural constituent of the DNA of a number of Bacillis subtilis phages (15)
  • glucose acceptors for these transferase enzymes.
  • glucose of glucosylated DNA does not interfere with nucleic acid hybridizations. That is, glucosylated DNA is frequently used in nucleic acid hybridizations (16) and has never been reported to interfere with the hybridization process. It should also be pointed out that glucose could be added enzymatically to the (5'-hydroxymethylcytosine-containing) probe molecules after they have formed hybrids with the target nucleic acid sequences. That is, the glucosyl transferase enzymes could be employed after hybridization of probe and target sequences as well as during preparation of the probe molecules. This order of operations might be preferable for situations where the probe molecules are produced chemically. This is because chemically produced polynucleotides are single stranded and because most of the known glucosyl transferase enzymes are significantly less active on single stranded DNA than on double stranded DNA (17,18).
  • glucosyl transferase enzymes Three types of glucosyl transferase enzymes were identified and partially characterised in 1961 and 1962 (16,17,18). Recently, most of the genes encoding these enzymes have been cloned (19,20), and some of them have been expressed at high levels in E. coli (19). These enzymes are encoded in the bacteriophages genomes. With two types of these enzymes a monosaccharide is produced: single glucose molecules are covalently attached to the 5' position of the grlucose-accepting base, hydroxymethylcytosine inaan aipha- or in a beta-O-glycosidic linkage.
  • a disaccharide is produced: a single glucose as attached in a beta-linkage on the number six carbon of a glucose that is already attached to the DNA.
  • This disaccharide is gentiobiose (21). All of these enzymes are active in. vitro and have been used to produce glucosylated DNA whose glucosylation pattern is different from the in vitro pattern on T-even bacteriophage DNA's (11,18).
  • the critical consideration for the present invention is that these enzymes can glucosylate, in vitro, any DNA sequence that contains the glucose-accepting base, HMC. That is, in vitro, these enzymes will glucosylate DNA other than T-even bacteriophage DNA if this DNA contains the potential glucose accepting nucleotide.
  • glucosylated DNA of any desired sequence can be produced: in vitro.
  • glucosyl transferase enzymes are welll known, they are not frequently used in the scientific community. They have been only partially characterized (20) and they have not been the subject of any studies designed to alter or modify the specificity of their enzymatic activity. In particular, it is not known if any of these enzymes or a mutationally altered form of any of these enzymes, might, under some conditions add a sugar other than glucose to DNA containing a 5' hydroxymethyl-cytosine base. That is, it is not presently known if the sugar donor molecule can be something other than UDP-glucose (11).
  • sugars other than glucose can be added.
  • glucosylated DNA by the action of one or more of the innumerable enzymes that are normally involved in producing or degrading oligo and poly -saccharides.
  • lactose synthetase and the galactosyl transferase subunit of lactose synthetase by itself can transfer galactose from the galactose donor molecule, UDP-galactose, to the disaccharide gentiobiose.
  • ⁇ -galactosidase which normally is involved in removing galactose residues from oligo and polysaccharides, can also add, galactose to a wide variety of sugars (and other -OH containing compounds) either by condensation or by acting as a transferase (23).
  • sugars other than glucose can be readily added to the glucose of glucosylated DNA by any of several enzymes that are not normally involved with nucleic acids.
  • galactose is covalently linked to DNA
  • the essential principle of this invention can be performed enzymatically by the use of galactose oxidase (24) to generate an aldehyde group attached to the galactose ring. If only a single galactose were present, a monoaldehydic polynucleotide could be produced.
  • the nucleic acid material used in performing this invention is not limited to glucosylated nucleic acids but also includes nucleic acids that contain non-backbone sugars other than glucose, nucleic acids containing carbohydrate chains, polyamino acid chains etc, or combinations thereof.
  • glucose moiety of glucosylated DNA can be readily and specifically oxidized by periodate.
  • the invention is not, of course, limited to the periodate technique.
  • the present invention shows for the first time that the glucose on glucosylated DNA can be a site for the covalent attachment of a variety of entities to a polynucleotide chain.
  • the glucose of glucosylated DNA has previously been covalently linked to compounds used to determine the presence of glucose on the DNA, this has been done only under conditions that destroy the polynucleotide chain or release the glucose from the base, hydroxymethylcytosine (see for example, reference 25).
  • this invention is based on the first and surprising realisation of a simple, non-destructive means of providing reactive attachment sites in large numbers on polynucleotides.
  • Glucosylated DNA can be oxidized by periodate, as already indicated, and the oxidized product is also chemically reactive as shown by its ability to form stable adducts with a variety of chemical compounds. Molecules that have never before been covalently attached to DNA can be attached to glucosylated DNA that has been oxidised (DNA ox ).
  • DNA was isolated by standared methods of phenol extraction from purified T4 phage particles.
  • the phage were purified by the well known technique of alternate cycles of high and low speed centrifugation.
  • T4 DNA that does not contain glucose was obtained by methods well known in the art.
  • a multiple mutant phage stock (gene 56 amE10, gene 42 amN55, denA S112, denB Sa 9 and ale (TBI) ) was grown for one cycle of infection in the non-permissive host E. coli B E and for a second cycle of infection in the non-suppressing host E. coli 834 as described (26).
  • This DNA contains low levels of glucose because most of the HMC bases, to which the glucose is enzymatically attached, have been replaced by cytosine.
  • This DNA will be referred to as T4 dC DNA or as non-glucosylated DNA.
  • T4 DNA will contain the unusual base HMC and have glucose residues attached to these bases.
  • This T4 glucosylated DNA will be referred to as HMC DNA.
  • HMC DNA This T4 glucosylated DNA
  • HMC and dC DNA's were either sonicated five times for 30 seconds at maximum power with a micro tip in a Branson sonicator, model W185D, or digested with the restriction endonuclease Taql (Boehringer Mannheim) at 65° in T4 buffer (33mM Tris-Acetate, pH7.9; 66mM Potassium Acetate; 10mM Mg-Acetate; 0.5mM DTT and 100 ⁇ g nuclease free BSA). Both of these treatments reduce the size of the DNA and the viscosity of the DNA solutions.
  • the DNA solutions were then ethanol precipitated and resuspended in either water or 50mM NaCl at 0.2 to 2.0mg/ml. Wild type T4 phage, multiple mutant phage suitable for making isogenic glucosylated and non-glucosylated T4 DNA as well as suitable E. coli host strains can be readily obtained from any number of sources well known in the art.
  • Periodate oxidation of nucleic acids is not new. Previously, the periodate oxidation of the sugar moiety of RNA molecules, but not of DNA moecules has been reported in the scientific literature. The ribose moiety at the 3' end of tRNA molecules uncharged with amino acids provide a structure (a cis-diol) that has been the object of periodate oxidation to a dialdehyde since at least 1960 (27). These oxidized tRNA molecules have been reacted with a number of reagents. See (5) for a review of this literature up to 1979. RNA molecules carrying reporter groups attached to these periodate produced aldehydes have been used as probes to locate the cellular sites of complementary DNA sequences (28).
  • RNA oxidisable group
  • glucose oxidisable group
  • the present demonstration of the use of periodate to oxidize nucleic acids is new in at least five ways.
  • the oxidizable group is glucose, not ribose.
  • the oxidizable group can be located at any position along the polynucleic acid chain instead of only at the 3' end.
  • the oxidization of a sugar moiety on a naturally occuring DNA is new.
  • the production of more than a single pair of aldehydes on a polynucleotide chain (of either RNA or DNA) by oxidization of sugar moieties is new and the oxidization of a sugar moiety that is not part of the sugar-phosphate backbone of a polynucleotide chain is new.
  • 2,4-dinitrophenylhydrazine (2,4-DNPH) is a well known chemical reagent specific for aldehyde and ketone groups.
  • This reagent has been used to detect the dialdehydes formed by periodate oxidation of the 3' ends of tRNA molecules (27). Essentially this procedure may be used to detect the aldehyde groups produced by periodate treatment of glucosylated DNA. The procedure may be as follows:
  • HMC glucosylated
  • Glucosylated DNA oxidized by periodate can undergo a variety of additional chemical reactions.
  • the reaction of DNA ox with 2,4-DNPH has been presented above.
  • the use of DNA ox to form Schiff bases that can be reduced with cyanoborohyride is now described.
  • Convenient conditions for producing Schiff bases with glucosylate DNA and their reduction by cyanoborohydride are 5 volumes of periodate treated DNA (at 100 to 2000 ⁇ g/ml in 50 mM NaCl). 1 volume of 10% acetic acid (adjusted to pH 3.5 with
  • Aromatic amine compounds are particularly useful for this purpose since they are more nucleophilic than aliphatic ones and, because of thier characteristic pK's of protonation, one can obtain a large measure of selectivity relative to aliphatic groups (30).
  • Tritiated cyanoborohydride (Amersham TRK.708) was diluted 100-fold with unlabelled 3mM cyanoborohydride to a specific activity of 34 mCi/mM.
  • the input radioactivity for the experimental data presented in Table 2 was from 5.8-7.0 x 10 4 cpm.
  • the glucosylated and non-glucosylated DNA's are the same as those used for the experiment described in Table 1.
  • the DNA was first digested with the restriction endonuciease Taq 1 (as described above), phenol extracted, ethanol precipitated and resuspended in 50 mM NaCl.
  • One portion of the digested DNA was oxidized with periodate as described above and then both oxidized and non-oxidized DNA's were mixed with m-aminobenzoic acid in the presence of cyanoborohydride as described above.
  • columns (a) and (b) are DNAox and columns (c) and (d) are glucosylated DNA that was not treated with periodate) and double stranded DNA size markers (columns (i) - (1): (i)4.3 and 3.6 kilobases (kb); (j) 5.4, 1.4 and 1.1 kb; (k) 4.0, 2.3 and 1.3 kb; (1) 3.2, 2.7 and 1.7 kb.
  • the faint band near the top of column (1) is a partial digest product. Examination of the figure shows that the band pattern in each of the taq 1 digests (columns (a)-(h)) is similar.
  • the individual bands migrate more slowly (and thus have a greater mass) than the individual bands from either of the other two reaction mixtures containing DNA ox (compare column (e) to columns (f) and (g) ).
  • the slower migration occurs only if both m-aminobenzoic acid and cyanoborohydride are present (column (e) ). If m-aminobenzoic acid is omitted from the reaction (column (f) ) or if cyanoborohydride is omitted from the reaction (column (g) ) the reaction products have the same mobilities as unreacted DNA ox (columns (a) and (b) ).
  • the molecular weight of m-aminobenzoic acid is 137.1. If one molecule of this compound was attached to a glucose on glucosylated DNA, the mass of that GC base pair (actually a G-HMC base pair since the glucose is attached to the HMC bases) would be increased by 12%.
  • Polynucleotides attached to complex compounds like mF can undoubtedly be produced much more easily by direct chemical coupling to polyaldehydic DNA than by incorporation of a bulky, complex nucleotide into a polynucleotide chain by either nick-translation or chemical synthesis.
  • polyaldehydic DNA allows one rapidly to synthesize novel polynucleotide derivatives and to test them for their behaviour under particular experimental conditions.
  • European Patent Specification No. 0243929 describes mF and related compounds which may be used in this invention.
  • glucosylated DNA can be oxidized by a simple chemical method to produce aldehyde groups which are highly reactive under appropriate conditions.
  • the production of aldehyde groups on glucosylated DNA by periodate treatment was demonstrated by reacting the oxidized DNA with the aldehyde-specific reagent 2,4-dinitrophenylhydrazine.
  • the reactivity of the aldehyde groups on DNA ox was demonstrated by their reaction with 2,4-DNPH and by the formation of Schiff bases with m-aminobenzoic acid and the reduction of these Schiff bases with cyanoborohydride.
  • the evidence presented suggests that the aldehydes so produced can be used to form stable adducts.
  • the chemical methods used for these demonstrations are easy to perform and can readily be scaled up. Other kinds of adducts can also be formed.
  • probes made from DNAox-adducts in nucleic acid hybridization reactions, will encounter the same problems that limit the sensitivity and usefulness of other kinds of probe-reporter complexes.
  • DNAox molecules can hybridize with a normal or only moderately reduced efficiency.
  • probes made from DNAox will encounter fewer of the problems referred to above than probes made from DNAox- reporter complexes and hence will allow the achievement of greater sensitivity than probes already linked to a reporter group.
  • glucosylated DNA hybridizes the glucosylated probes to the target sequences and then oxidize the non-backbone sugar and add the reporter groups.
  • This sequence of events would, require at least one additional manipulation by the end user.
  • this approach would often not be desirable when the target nucleic acid is RNA because the riboses at the 3' ends of the RNA chains could also be oxidized (at least by periodate) and linked to the reporter groups.
  • the data in table 3 is from an experiment in which 32 P-RNA was hybridized for 17 hours at 66-67°C in 2 ml 2 ⁇ SSC with polyaldehedic and wild-type (glucosylated) T4 DNA that had been loaded onto nitrocellulose filters as described (16), except that the T4 DNA was fixed to the filters by UV treatment (33) instead of baking at 80°C under vacuum.
  • the 32 P-RNA (40,000 cpm in 2 ⁇ l; contains 327 bases of T4 and about 100 bases of non T4 DNA) was prepared as a Riboprobe, by standard proceedures, from a clone of T4 DNA coming from the gene 32 region (approximate kilobase co-ordinates of 146.25 to 146.5 on the standard T4 map).
  • each hybridization vial done in duplicate, there were three filters; one was charged with 0.8 ug of plasmid pBR322 DNA and the other two were charged with equal amounts of either polyaldehydic or wild-type T4 DNA.
  • the filters were batch washed at room temperature: first two times for at least 30 minutes in 2 ⁇ SSC, then once for at least 15 minutes in
  • aldehydes might interact with the NH 2 groups on adenine, guanine or cytosine bases and thus might, a priori, be expected to reduce hybridization efficiencies. In any case, this does not have to occur to a significant extent and consequently may have important practical uses.
  • RNA-DNA hybridization experiment (table 4) increasing amounts of 32P -RNA (1-10 ⁇ l of the same Riboprobe preparation as used above) were hybridized, in solution (66°C for 18 hours in 1 ml containing 800 ⁇ l of 2 ⁇ SSC and 200 ⁇ l of the denatured DNA in essentially 4 ⁇ SSC (16)), with a constant amount of either wild-type T4 DNA (12.9 ug) o ⁇ r polyaldehydic T4 DNA (11.0 ug) that had been heat denatured in alkali as described previously (16).
  • RNA-DNA hybrid formation was determined by measuring the ⁇ fraction of the input counts that became resistant to RNase digestion (to 100 ⁇ l of hybridization solution, 1 ug of Yeast tRNA and 5 ug of pancreatic RNase were added, incubated for 45 minutes at room temperature and acid precipitated).
  • 3 2 P-RNA is RNase resistant.
  • T4 DNA 14 C-labelled T4 DNA (wild type) was prepared from phage particles by standard methods. Samples of this DNA was oxidized as described above. The results are tabulated in table 5 below. The absolute efficiency of the assay (25, 15 %, see last line of the tabulation) is satisfactory, and therefore the relative efficiencies given in the first line of the tabulation can be taken to be valid.
  • the hybridization method was essentially that of Mattson et al. (1983) J. Mol. Biol. 170343-355, except that water was used in place of formamide and the hybridizations were done at 66°C instead of 42°C.
  • Each 1ml of the hybridization solution is prepared by first mixing 1-10 microlitres 14 C-DNA solution (the actual colume is chosen so as to give the required number of counts) with 0.25 ml of solution TSE (see above) plus 0.25ml of 1M NaOH.
  • the solution is then ready to be transferred to the hybridization vials (glass, siliconized). Each vial receives 2ml of the final mixture.
  • Nitrocellulose filters were charged with an excess of single-stranded DNA according to the method of Mattson et al. 1983 (loc. cit.). Three types of filter were prepared: T4 DNA (unmodified) 8 microgrammes/ug/filter), calf thymus DNA (4 microgrammes/filter), and salmon-sperm DNA (4 microgrammes/filter). The filters were pre-treated in a mixture of 10 volumes of SSC (double strength) and 1 volume Denhardt solution (fifty-fold strength) for at least 2h. at 66° prior to hybridization.
  • More than one filter can be put into each hybridization vial. Before they were placed in the vials the filters were coded with a soft-lead pencil and wetted with SSC (six-fold strength). The coding is done so that the filters need not be washed separately, and the wetting is carried out in order to be able to reject those few filters that do not wet satisfactorily.
  • Hybridization took place overnight at 66° in a shaking water bath.
  • the filters were then washed all together in copious quantities of SSC (double strength) at room temperature for 30 minutes. They were then rinsed once in water, transferred to scintillation vials, dried, and counted.
  • T4-HMC Hydroxymethylcytosyl T4 DNA
  • Tag 1 labelled with 32 P
  • a portion of the labelled DNA was oxidized, and reacted with biotin amidocaproyl hydrazide (BioHZ) (Sigma Chemical) to produce a biotin derivative of T4 (T4-Bio).
  • BioHZ biotin amidocaproyl hydrazide
  • T4-HMC was digested with Taq l (2.5 U/ug) at 65 C, precipitated with isopropanol and sodium acetate, and resuspended at 1 ug/ul in TE (TE: 10 mM Tris pH 8.0, 0.1 mM EDTA. A portion of this DNA was adjusted to 20 mM Tris with 1 M Tris pH 9.5, and the 5' phosphate groups removed by incubation at 37 C for 30 minutes with bacterial alkaline phosphatase. After the incubation, the enzyme was removed by extraction with phenol and chloroform-isoamyl alcohol (24:1), and precipitated as above.
  • the DNA was resuspended at 1 ug/ul in TE.
  • the 5' -dephosphorylated DNA was incubated at 37 C for 30 minutes with gamma [ 32 P]ATP and T4 polynucleotide kinase under standard conditions [36], precipitated as above, washed with 70% ethanol, and resuspended at 1 ug/ul in water.
  • 15 ul of the 3 2 P-labelled T4-HMC was oxidized for 30 minutes with 60 mM sodium periodate and 50 mM sodium acetate pH 5.6.
  • the oxidized DNA (T4-OX) was precipitated and washed as above, then resuspended to around 0.5 ug/ul in 1% acetic acid pH 3.5.
  • the T4-OX was reacted overnight with an equal volume of 27 mM BioHZ (dissolved in 50% acetonitrile, l%> acetic acid pH 3.5). After the reaction, the
  • T4-Bio was precipitated and washed, and suspended in TES (10 mM Tris pH 8.0, ImM EDTA, 1% SDS) .
  • Amersham Hybond-N nylon filters were wetted, soaked in 20 ⁇ SSC (Maniatis et al. 1982), and air-dried.
  • Aqueous solutions at 1 ug/ul were prepared from Taq 1-digested T4-HMC and sheared salmon-sperm DNA. These were denatured by boiling for 5 minutes, and then 5 ul lots were deposited in dots on the prepared filters (as two applications of 2.5 ul) : sufficient 5 ug dots were prepared for hybridization to each probe in triplicate.
  • the filters were air-dried, and the DNA was fixed to the filters by UV-irradiation. Filters carrying "no DNA" weres also prepared.
  • the filters were prehybridized at 42 C overnight, in 5 ml of 6 ⁇ SSC, 50% deionized formamide, 50 ug/ul salmon-sperm DNA, 0.1% SDS, 2 ⁇ Denhardt's solution (50 ⁇ Denhardt's: 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin).
  • T4 DNA probes T4-HMC and T4-Bio
  • Hybridization was at 42 C for 8 hours, with constant agitation.
  • the filters were rinsed at ambient temperature in 2 ⁇ SSC, then washed at 50 C for 15 minutes in 0.2 ⁇ SSC, 0.1% SDS. Filter-bound radioactivity was determined by Cerenkov counting.
  • Table 6 shows the filter-bound counts expressed relative to (100% + amount of 32 P-T4-HMC bound to T4-HMC), after subtraction of background counts, and normalization to a constant number of counts added to each hybridization mix.
  • the raw data from which the above figures was derived are shown in table 7.
  • the recorded cpms are the means of triplicates.
  • the figures in parentheses are normalized to 10 6 cpm added to each hybridization (the actual cpm added were: T4-HMC 947400; T4-Bio 998100).
  • the T4-Bio probe has a similar melting profile to T4-HMC, suggesting that reduced stability of the duplex is not responsible for the lower binding of the derivative.
  • the T4-Bio DNA hybridizes about one-fifth as efficiently as native T4-HMC (21.7%), and the T4-Bio derivative has a similar melting profile to T4-HMC, implying that the modified DNAs do not form less stable duplexes.
  • polyaldehydic DNA all by itself, is the DNA probe of choice.
  • Polyaldehydic DNA is the probe of choice because it can hybridize efficiently to target sequences, thus allowing virtually any reporter group, whatsoever, to be added to a probetarget hybrid molecule.
  • the preferred method of practicing the invention in the field of DNA-based diagnostics would be to produce glucosylated DNA probe sequences in vivo, in E. coli by employing, as described in (31), Bacteriophage T4 (T4) denB mutant phage, in conjunction with plasmids containing both a region of homology with T4 and the (preprobe) sequence or sequences to be converted into glucosylated or polyaldehydic probes.
  • T4 Bacteriophage T4
  • T4 denB mutant phage in conjunction with plasmids containing both a region of homology with T4 and the (preprobe) sequence or sequences to be converted into glucosylated or polyaldehydic probes.
  • the in vivo produced glucosylated probes can be of any length, it is preferred to introduce recognition sites for the restriction endonuclease Taq 1 (one of the few restriction endonucleases that can efficiently cut glucosylated DNA) at regular intervals into the preprobe sequence, say every 50-100 base pairs, in order to (1) facilitate the purification of the glucosylated probes away from the T4 sequences, by hybridization to complimentary single stranded sequences fixed to a solid support followed by elution of the now purified glucosylated probes, and (2) in order to have short probes that exhibit favourable hybridization kinetics with target sequences.
  • Taq 1 one of the few restriction endonucleases that can efficiently cut glucosylated DNA
  • reporter groups e.g. biotin
  • Attachment of reporter groups to polyaldehydic probes after hybridization to the target sequences provides better opportunities to increase the signal to noise ratio.
  • reporter groups e.g. biotin
  • the target nucleic acid is RNA
  • to hybridize with polyaldehydic probes because oxidization after hybridization could reduce the signal to noise ratio, a consequence of the oxidization of the ribose moiety at the 3' end of the RNA target molecules.
  • Clearly probes produced according to this invention allow a large degree of flexibility for the order in which the operations can be preformed.
  • both the chemically reactive aldehyde groups and the reporter groups can be introduced either before or after the hybridization step.
  • Polyaldehydic probes can readily be employed with currently available detection systems such as avidin-biotin detection systems and immunological detection systems (i.e. an antigenic entity could be linked to the aldehyde group). Polyaldehydic probes also create the possibility of employing new detection systems, for example, a component of a light generating system, such as a luciferase-like enzyme, could be attached to the aldehyde groups.

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Abstract

La présente invention se rapporte à une sonde utilisant des polynucléotides et comprenant une séquence de base transformable par hybridation en un polynucléotide présélectionné ainsi qu'une multitude de groupes rapporteurs reliés chacun à la séquence par l'intermédiaire d'un groupe aldéhyde, lequel a été obtenu par oxydation d'un résidu de sucre sans ossature. L'utilisation de groupes d'aldéhydes permet d'effectuer aisément un marquage après hybridation qui offre des avantages importants dans le domaine des analyses/diagnostics.
PCT/GB1988/000754 1987-09-17 1988-09-16 Polynucleotides polyaldehydiques utilises comme sondes, leur preparation et utilisation WO1989002475A1 (fr)

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Cited By (7)

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EP0571908A2 (fr) * 1992-05-29 1993-12-01 Eastman Kodak Company Dispositif et méthode de génération de signal vidéo entrelaçé utilisant un capteur d'image haute résolution à balayage progressif
DE19850594A1 (de) * 1998-11-03 2000-05-04 Biochip Technologies Gmbh Verfahren zur Markierung von Nukleinsäuren
WO2006117161A3 (fr) * 2005-05-02 2007-01-11 Basf Ag Nouvelles strategies d'etiquetage pour detection sensible d'analytes
US7910335B2 (en) 2005-10-27 2011-03-22 President And Fellows Of Harvard College Methods and compositions for labeling nucleic acids
US8193335B2 (en) 2006-10-31 2012-06-05 Baseclick Gmbh Click chemistry for the production of reporter molecules
KR101335218B1 (ko) 2005-05-02 2013-12-12 바스프 에스이 분석물의 감응성 검출을 위한 신규한 표지화 전략
US10138510B2 (en) 2008-05-16 2018-11-27 Life Technologies Corporation Dual labeling methods for measuring cellular proliferation

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EP0133473A2 (fr) * 1983-07-05 1985-02-27 Enzo Biochem, Inc. Marquage in vivo de séquences nucléotides
EP0184056A2 (fr) * 1984-11-27 1986-06-11 Molecular Diagnostics, Inc. Production d'ADN à grande échelle
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EP0133473A2 (fr) * 1983-07-05 1985-02-27 Enzo Biochem, Inc. Marquage in vivo de séquences nucléotides
EP0184056A2 (fr) * 1984-11-27 1986-06-11 Molecular Diagnostics, Inc. Production d'ADN à grande échelle
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EP0212546A2 (fr) * 1985-08-13 1987-03-04 Enzo Biochem, Inc. Procédé pour l'étiquetage des séquences polynucléotidiques

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0571908A2 (fr) * 1992-05-29 1993-12-01 Eastman Kodak Company Dispositif et méthode de génération de signal vidéo entrelaçé utilisant un capteur d'image haute résolution à balayage progressif
EP0571908A3 (en) * 1992-05-29 1993-12-15 Eastman Kodak Co Apparatus and method for generating an interlaced video signal using a progressively scanned high resolution image sensor
DE19850594A1 (de) * 1998-11-03 2000-05-04 Biochip Technologies Gmbh Verfahren zur Markierung von Nukleinsäuren
JP4944098B2 (ja) * 2005-05-02 2012-05-30 ビーエーエスエフ ソシエタス・ヨーロピア 分析物の高感度検出のための新規標識方法
AU2006243370B2 (en) * 2005-05-02 2012-06-28 Basf Aktiengesellschaft New labelling strategies for the sensitive detection of analytes
EP2256126A1 (fr) * 2005-05-02 2010-12-01 baseclick GmbH Stratégies de marquage pour la détection sensible d'analytes
US9005892B2 (en) 2005-05-02 2015-04-14 Baseclick Gmbh Labelling strategies for the sensitive detection of analytes
US8129315B2 (en) 2005-05-02 2012-03-06 Baseclick Gmbh Labelling strategies for the sensitive detection of analytes
WO2006117161A3 (fr) * 2005-05-02 2007-01-11 Basf Ag Nouvelles strategies d'etiquetage pour detection sensible d'analytes
KR101335218B1 (ko) 2005-05-02 2013-12-12 바스프 에스이 분석물의 감응성 검출을 위한 신규한 표지화 전략
JP2008539703A (ja) * 2005-05-02 2008-11-20 ビーエーエスエフ ソシエタス・ヨーロピア 分析物の高感度検出のための新規標識方法
TWI406952B (zh) * 2005-05-02 2013-09-01 Basf Ag 分析物之敏感偵測的新標記策略
US8859753B2 (en) 2005-10-27 2014-10-14 President And Fellows Of Harvard College Methods and compositions for labeling nucleic acids
US7910335B2 (en) 2005-10-27 2011-03-22 President And Fellows Of Harvard College Methods and compositions for labeling nucleic acids
US9790541B2 (en) 2005-10-27 2017-10-17 President And Fellows Of Harvard College Methods and compositions for labeling nucleic acids
US8193335B2 (en) 2006-10-31 2012-06-05 Baseclick Gmbh Click chemistry for the production of reporter molecules
US10138510B2 (en) 2008-05-16 2018-11-27 Life Technologies Corporation Dual labeling methods for measuring cellular proliferation

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