Patent Application
20100081801
The invention is related to a method for determining an unknown PNA sequence information and to methods for the selection, identification, accumulation and amplification of PNA molecules or producing PNA molecules capable of forming PNA/target molecule complexes.
Single stranded nucleic acids like RNA or ssDNA are capable of forming a three-dimensional structure comparable with folding of proteins, which is essentially determined by the nucleic acid sequence. These three-dimensional structure are based mainly on intramolecular base pair interactions and promote the capability to recognize the surface structure of target molecules and to bind to target molecules having surface structures matching with the three-dimensional structure of the nucleic acid. Such nucleic acids capable of binding to target molecules due to three-dimensional matching are called aptamers. The target molecules may be of any species like small organic molecules (e.g. coffein), large organic molecules (e.g. synthetic polymers), peptides, proteins, enzymes, amino acids, saccharides, nucleotides, oligo- and polynucleotides, hormones, polysaccharides, or surface structures of cells or viruses. Aptamers may even be capable of distinguishing between enantiomers and recognizing the presence or absence of functional groups like methyl- or hydroxyl groups. Accordingly, aptamers may be used in particular in therapeutic and/or diagnostic applications (Jayasena, S. D., Aptamers: An emerging class of molecules that rival antibodies in diagnostics, Clin. Chem. 1999, 45(9), 1628-50; Herman, T. and Patel, D. J., Adaptive recognition by nucleic acid aptamers, Science 2000, 287(5454), 820-5).
In order to identify aptamers capable of binding to a defined target molecule it is known to apply selection methods based on providing a nucleic acid library, which is then contacted with target molecules of a defined target molecule species. Binding nucleic acids are amplified and accumulated until the concentration of binding nucleic acids is sufficient for cloning and/or sequencing. With the sequence information obtained the aptamers for the defined target molecule species may be generated in production scale and used for the intended application. One such method is the so-called SELEX method described in the publications Tuerk, C., Gold, L., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science 1990, 249(4968), 505-10 and Ellington, A. D. and Szostak, J. W., In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346(6287), 818-22.
Peptide nucleic acids (PNA) are synthetic molecules which are capable of hybridizing with oligomeric or polymeric nucleic acids. PNA molecules resemble such nucleic acids but differ in the backbone chain being constituted of a peptidic achiral structure based on N-(2-aminoethyl)glycine monomers instead of ribose-phosphate monomers. The natural bases are thereby attached to the backbone chain by carboxymethylene units. Remarkably, the backbone chain of PNA molecules is electrically neutral, whereas the backbone chain of nucleic acids is charged. Further, PNA molecules are resistent against degradation by nucleases, proteases or peptidases. Finally, PNA molecules have shown a comparatively low toxicity in biological systems, if any. Additional reference is made to the publication McMahon, B. M. et al., Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration, Antisense Nucleic Acid Drug Dev. 2002, 12(2), 65-70.
Accordingly, it would be favorable to obtain and identify PNA molecule species, which function like aptamers, in particular for therapeutic and/or diagnostic applications or which provide other certain properties including but not limited to catalytic functions, since PNA molecules are highly stable in biological environments and lack a charge which makes a number of target molecules accessible for PNA aptamers that are eluded from selection processes involving natural nucleic acids due to unspecific binding based on electrostatic interactions. One possibility to obtain such a PNA molecule species with binding properties or other functional properties would be to bring a library with PNA molecules of a plurality of different PNA molecule species in contact with a desired target molecule and separate the unbound PNA from the PNA/target molecule complexes. One major problem herein is the identification of obtained PNA molecules with the desired properties, since the amount of PNA will not be sufficient for analysis by weight measurement or structure determination using standard methods (e.g. mass spectrometry or spectroscopy). Therefore the selected PNA molecule species need to be amplified prior to analysis. However, presently there are no technologies existing providing means for the amplification of PNA or their sequencing. Because of this reason methods like SELEX are not applicable for PNA hence they involve an amplification of the library components.
Attempts to transfer of PNA sequence information to natural nucleic acids have been reported in Schmidt, J. G., Information transfer from peptide nucleic acids to RNA by template-directed synthesis, Nucleic Acids Res. 1997, 25(23), 4797-802. However the low efficiency of these chemical coupling procedures make these methods unapplicable to practical processes. As a result these technology has been pursued during the last years, because there is an urgent need in the field for a method that allows amplification of PNA with reasonable efficiency within an acceptable period of time. Furthermore there is need in the field for a process to select, amplify and sequence PNA molecules which function like aptamers or provide other functional properties.
It is known in the art how to sequence nucleic acids (Sanger F. et al., DNA-sequencing with chain-terminating inhibitors, Proc. Nat. Acad. Sci. 1977, 74, 5463-5467 and Hunkapiller, T. et al., Large-scale and automated DNA sequence determination, Science 1991, 254, 59-67). However these processes are not transferable to PNA due to the different backbone chain chemistry.
Synthesis of PNA per se is further known by using amplified DNA as template (Rosenbaum, D. M. and Liu, D. R., Efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid aldehydes. J. Am. Chem. Soc. 2003, 125(46), 13924-5) or by sequencing the obtained nucleic acid bearing the PNA sequence information and subsequent chemical synthesis of PNA by standard methods (Christensen, L. et al., Solid-phase synthesis of peptide nucleic acids, J. Pept. Sci. 1995, 1(3), 175-83 and Thomson, S. A. et al., Fmoc mediated synthesis of peptide nucleic acids, Tetrahedron 1995, 51, 6179-94).
A first object of the invention is to provide means for sequencing PNA oligomers or polymers.
A further object of the invention is to provide means to select and determine the sequence of PNA oligomers or polymers capable of binding a defined target molecule.
A further object of the invention is to provide means for identifying, accumulating and/or producing PNA molecules capable of binding to a defined target molecule.
A further object of the invention is to provide means for effectively separating unbound PNA molecules from PNA/target molecule complexes.
A further object of the invention is to provide PNA molecules, which bind with high affinity to defined target molecules.
For achieving the first mentioned object, the invention provides a method for determining the sequence of PNA molecules of a specific PNA molecule species, wherein PNA molecules are contacted with one or several different nucleic acid molecule species comprising nucleic acid molecules with at least one nucleotide, wherein the nucleic acid molecules at least partially comprise a nucleic acid sequence that is complementary to at least a partial sequence of the PNA molecule, wherein nucleic acid molecules having complementary sequences bind to the PNA molecules forming nucleic acid/PNA hybrids, wherein nucleic acid molecules with non-complementary sequences are separated from the nucleic acid/PNA hybrids, wherein nucleic acid molecules with non-complementary sequences are optionally degraded enzymatically, wherein thereafter the nucleic acid/PNA hybrids are dissociated into single stranded nucleic acid molecules and PNA molecules, wherein the single stranded nucleic acid molecules are subjected to a sequencing process providing sequence information about the single stranded nucleic acid sequence, and wherein the sequence information is optionally translated into the complementary PNA sequence information.
The invention is based on the fact that PNA may hybridize with nucleic acids with high affinity and stringency and that hybridizing nucleic acids may be sequenced using standard processes, thereby obtaining sequence information being complementary to the sequence of the PNA.
The sequence length of the PNA may be at least 5, preferably at least 10, more preferably at least 15.
The sequence length of the nucleic acid molecules may be at least 2, preferably at least 3, more preferably at least 4. The method of the invention can be carried out in a variety of embodiments which differ basically in the sequence length of the nucleic acid molecules contacted with the unknown PNA molecule.
In one embodiment the sequence length of the nucleic acid molecules is at least the sequence length of the PNA molecules, wherein binding of complementary PNA molecules and nucleic acid molecules is carried out under hybridization conditions forming the nucleic acid/PNA hybrids without ligation of nucleic acid molecules bound to the PNA molecules. In this most simple embodiment the nucleic acid molecule may be longer than the PNA molecule, wherein the overhang sequences may e.g. be used for amplification of the nucleic acid molecules after dissociation, for immobilization or for detection using standard routines.
The invention may further comprise means for specific enzymatical degradation of non-hybridized oligonucleotides. Examples for the enzymatical degradation are the use of nucleases (e.g. DNaseI), use of single-strand specific nucleases (e.g. but not restricted to S1 nuclease and Mungobean nuclease), use of restriction enzymes either of type II (e.g. XbaI, HindIII etc.) or of type IIs (e.g. MlyI, BseRI etc.). Within the following examples further details of some of the degradation processes may be taken.
In practice it will be favorable to first obtain or create a nucleic acid library, wherein the nucleic acid molecules or partial sequences thereof are randomized. The randomized part should have a length corresponding to the sequence length of the PNA under investigation (this is either known, since the PNA may originate from a PNA library, or may be determined using standard methods for molecular weight measurement).
In another embodiment the sequence length of the PNA molecules is greater than a total sequence length or a randomized partial sequence length of the nucleic acid molecules, preferably by a factor of 2 or more, more preferably by a factor of 3 or more, most preferably by a factor of 4 or more, wherein nucleic acid molecules binding adjacent to each other to the PNA molecules are ligated chemically or enzymatically, preferably enzymatically, forming the nucleic acid/PNA hybrid. Within this embodiment the total sequence length of the nucleic acid molecules may be larger that the sequence length of the PNA provided that the randomized sequence length is as above. If the randomized sequence length is one half or more than one half of the sequence length of the PNA molecules, then typically two different nucleic acid molecules of the randomized library will bind adjacent to each other. Then ligation must be performed between these two nucleic acid molecules. The overhangs may, again, be used for amplification, immobilization, detection, etc.
In another embodiment, the total sequence length of the nucleic acid molecules comprised in the randomized nucleic acid library may be less than the sequence length of the PNA, wherein the total sequence is randomized. Then two, three, four, five, six, seven, eight, nine, ten or more nucleic acid molecules will bind adjacent to each other to the PNA. Then n−1 ligation reactions are carried out (n=number of binding nucleic acid molecules) for making the hybrid.
In another embodiment, the total length of the nucleic acid molecules is one, i.e. the nucleic acid molecule library comprises a mixture of all nucleic acid monomers. Again n−1 ligation reaction must be carried out for making the hybrid.
The ligation reactions may be carried out with any process known in the art, although enzymatical ligation is preferred. Examples for chemical ligation reactions are the cyanogen bromide (CNBr) process and the 1-(3-[dimethylamino]propyl)-3-ethylcarbodiimide hydrochloride (EDC) process.
With respect to the enzymatic ligation, it is preferred to carry this out using a ligation enzyme selected from the group consisting of “DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, DNA ligase V, T4 DNA ligase, Tag DNA ligase, T4 RNA ligase, T4 RNA ligase II, ThermoPhage™ single-stranded DNA ligase, Rma DNA ligase, Tsc DNA ligase, E. coli DNA ligase, LdMNPV DNA ligase, LigTK, Mth ligase, PBCV-1 DNA ligase, Pfu DNA ligase, Sealase, T4 ATP ligase, Vaccinia ligase, Tfi DNA ligase, Tth DNA ligase, Band IV, DREL, gp24.1, P52, RM378 RNA ligase, TbMP52, Rcl1p, DNA ligase D, XRCC4-ligase, T7 DNA ligase, Bst ligase, DraRnl”, preferably using T4 RNA ligase. Within the following examples further details of some of the standard processes may be taken. Standard processes for enzymatic ligation reactions are well known to the skilled artisan and need not be described here in detail.
The basic process of selection and identification according to the invention includes the following steps:
If the amount of selected PNA molecules with binding affinity to the target molecule is too low for sequencing the selected PNA molecules are amplified prior to sequencing.
If the selection efficiency of the process is too low to sufficiently reduce the presence of PNA molecules which are not binding to the target molecule an additional round of selection can be performed as described in the following:
The invention further provides means to identify PNA molecules with certain properties like binding affinities or catalytic properties either after a single selection step and subsequent identification of selected PNA molecules wherein the sequence information from PNA is transferred to nucleic acids, RNA or DNA. This provides the possibility of amplifying the sequence information of the PNA by amplification of the obtained nucleic acid with standard methods (Mullis, K. B. and Faloona, F. A., Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction, Methods Enzymol. 1987, 155, 335-50) and their subsequent sequencing, whereas the obtained sequence can be translated to complementary PNA sequence information. This information can be used for a chemical de novo synthesis of PNA molecules capable of binding a defined target molecule or providing other properties.
Alternatively the amplified DNA can be used for synthesis of PNA molecules in a template directed manner which equals an amplification of PNA molecules for a subsequent selection round.
The invention may further comprise means for the separation of nucleic acid/PNA hybrids from non-hybridized nucleic acids in the order of magnitude of at least 10E4, preferably 10E6, more preferably 10E8, most preferably 10E10 by affinity based methods using affinity tags connected either to the PNA or to the nucleic acid, selected from the group consisting of e.g. “biotin-streptavidin system, strep-tag, digoxigenin, His-tag etc.” and a matrix selected from the group consisting of e.g. “agarose, sepharose, magnetic beads etc.”, carrying the corresponding binding partner for the affinity tag, wherein these methods also involve the use of specifically cleavable functional groups in the linker chains to the affinity tags selected from the group consisting of e.g. “reductive cleavable disulfide groups, photolabile groups, pH-sensitive groups etc.” for the specific elution of nucleic acid/PNA hybrids from the matrix.
The invention may further comprise means to separate nucleic acid/PNA hybrids from non-hybridized nucleic acids in the order of magnitude of at least 10E4, preferably 10E6, more preferably 10E8, most preferably 10E10 using chromatographic or electrophoretic methods selected from the group consisting of “HPLC, ion-chromatography, capillary electrophoresis, free flow electrophoresis, capillary gel electrophoresis, micellar electrokinetic capillary chromatography, capillary electrochromatography, non-gel-sieving, affinity-capillary-electrophoresis, capillary ion electrophoresis, HPLC, LC, ion-chromatography”.
The separation methods as well as the degradation methods can be combined with any methods for generating PNA complementary DNA mentioned above.
It will be favorable if the single stranded hybrid nucleic acid molecules are amplified, preferably using PCR, prior to the sequencing process.
The invention further comprises means for the amplification of generated complementary DNA by a variety of methods concerning different methods for generation of primer hybridization sites either by ligation of terminal linker oligonucleotides at the 5′- and/or the 3′-terminus of generated complementary nucleic acids, wherein methods are involved to reduce background arising by directly ligated linker oligonucleotides using restriction endonucleases, or by use of nucleic acid/PNA chimera wherein nucleic acids in the chimera are elongated by terminal nucleotidyltransferases to provide primer hybridization sites, or by use of nucleic acid/PNA chimera, wherein nucleic acids in the chimera form hairpin loops to provide start-oligonucleotides being elongated by nucleic acid fragments and ligation, wherein hairpin loops can be cut by restriction endonucleases or by single-strand specific nucleases taken out of the group consisting of “S1 nuclease, Mungobean nuclease etc.”. Within the following examples further details of some of the mentioned processes may be taken.
The invention further comprises a method for separating unbound PNA molecules from a solution comprising at least one target molecule species and PNA molecules of a plurality of different PNA molecule species, wherein PNA molecules of a PNA molecule species with a binding affinity to the target molecule species form PNA/target molecule complexes with the target molecules of this target molecule species, wherein the solution comprises an ionic compound effective to promote at least a partial electrical charge to either unbound PNA molecules or PNA/target molecule complexes, wherein the solution is subjected to an electrophoretic separation method comprising application of an electric field to the solution, and wherein PNA/target molecule complexes or unbound PNA molecules with a partial electrical charge promoted by the ionic compound obtain an electrical migration component of translation in the electrical field, thereby being separated from components in the solution having a different charge/size ratio or no electrical charge.
This aspect is based on the finding that ionic compounds in the solution comprising unbound PNA molecules and PNA/target molecule complexes are capable of associating with one of both groups only thereby imparting an electrical charge to the associated group. Typically, the ionic compound will promote at least a partial electrical charge to the PNA/target molecule complexes. This allows separation methods based on application of an electrical field, to which PNA molecules are normally not sensitive due to the neutral backbone chain, in contrast to nucleic acids.
The ionic compound may be in particular an ionic detergent, preferably an ionic detergent selected from the group consisting of “benzethonium-chloride, benzethonium-hydroxide, cetylpyridinium-bromide, cetylpyridinium-chloride, cetyltri-methylammonium-bromide, cetyltrimethylammonium-chloride, (2-hydroxyethyl)trimethylammonium salts, denatoniumbenzoates, denatoniumsaccharides, dodecyl-sulfates (preferably sodium-dodecyl-sulfate (SDS) but also lithium-dodecyl-sulfate and ammonium-dodecyl-sulfate), hexadecyltrimethylammoniumbromide (CTAB), hexadecyltrimethylammonium-chloride (CTAC), lauroylsarcosine, N,N-dimethyldecylamine-N-oxide (DDAO), N,N-dimethyldodecylamine-N-oxide (LDAO), sodium-bis(2-ethyl-hexyl)-sulfosuccinate, butanesulfonates, chenodeoxycholates, cholates, decanesulfonates, deoxycholates (DOC), sodium-docusate, dodecanesulfonates, glycocholates, glycodeoxy-cholates, heptanesulfonates, hexadecanesulfonates, octanesulfonates, octylsulfates, propanesulfonates, taurochenodeoxycholates, taurocholates, taurodehydrocholates, taurodeoxycholates, taurolithocholates, tauroursodeoxycholates, tetradecylsulfates, tert-octyl-phenyl-propane-sulfonic-acid (TOPPS), Triton X-100, 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS), 4′-amino-7-benzamido-taurocholic acid (BATC)” and preferably sodium dodecylsulfate (SDS), or a mono-, bi-, or tri-nucleotide taken from the group consisting of e.g. but not restricted to “adenosine, cytosine, guanine, thymine, uracile, desoxyadenosine, desoxycytosine, desoxy-guanine, desoxythymine”, or oligonucleotides respectively desoxyoligonucleotides with a length of five, preferably four, more preferably three, most preferably two monomers.
The separation method may be selected from the group consisting of “capillary electrophoresis, capillary gel electrophoresis, micellar electrokinetic capillary chromatography, capillary electrochromatography, non gel sieving, affinity capillary electrophoresis, capillary ion electrophoresis, HPLC, LC, ion chromatography”, preferably is capillary electrophoresis. For a review of electrophoretic separation methods and details of such methods, which are common in the art reference is made to e.g Righetti, P. G., Electrophoresis: the march of pennies the march of dimers, J. Chromatogr. A 2005, 1079(1-2), 24-40.
The concentration of the ionic compound in particular detergents may be below the Critical Micellary Concentration (CMC), preferably is below 8 mmol/l, more preferably is below 5 mmol/l, even preferably is in the range between 0.01-3 mmol/l, most preferably is in range between 0.1-1 mmol/l.
The invention further relates to PNA molecules obtainable by a method of the invention.
A target molecule species may be any species and is not restricted to the group consisting of “small organic molecules, peptides, proteins, enzymes, amino acids, hormones, polysaccharides, and surface structures of cells or viruses”. In particular, the target molecule species may as well be non-biological and comprise any molecular structure for which a need of detection exists, e.g. explosives, environmentally dangerous substances, and the like.
A PNA molecule consists of a backbone chain of at least two monomers, wherein to each of the monomers carries one nucleotide bases: adenine, cytosine, guanine, uracile or thymine. A PNA molecule is capable of hybridizing with a oligo- or polynucleic acid of natural structure. The monomers in one PNA molecule may be different or the same. Preferably the monomers are the same and N-(2-Aminoethyl)glycine, wherein the nucleotide bases are attached to the backbone chain via carboxymethylene units.
Unbound PNA molecules are molecules that are not bound to a target molecule. Preferably unbound PNA molecules are also not bound to any other compound present in the solution.
A molecule species is defined by a singular specific chemical structure. All molecules of a molecule species have the identical specific chemical structure. Preferably all molecules of a molecule species further have the same three-dimensional structure in space.
The molecules of different molecule species differ from each other in the three-dimensional structure and, optionally, in the chemical structure.
A PNA molecule binds to a target molecule with a binding affinity, if the three-dimensional structure of the PNA molecule fits to the three-dimensional structure of the target molecule of to a partial structure thereof. Typical affinity values are better than 10 μM.
A PNA/target molecule complex typically does not involve covalent bonds, but rather hydrogen bonds, electrostatic, hydrophobic and/or Van-der-Waals interactions.
The sequence length of a PNA molecule is defined as the number of backbone monomers present in the molecule.
Accumulation of a molecule species comprises increasing the concentration of the molecule species in a solution and/or increasing the number of the molecules of the species in a sample.
A PNA library comprises PNA molecules of a plurality of different PNA molecule species. The PNA molecule species typically differ in the sequence of the nucleotide bases attached to the backbone chain only.
Different nucleic acid species differ in the sequence of the nucleotide bases attached to the backbone chain.
Hybridization between PNA molecules and nucleic acid molecules takes place by hydrogen bonding between pairs of nucleotide bases present on the nucleic acid and the PNA if the sequences of the nucleotide bases of the PNA and of the nucleic acid match. Preferably the match is a complete and a 100% match to the shorter of both, if any.
The amplification of nucleic acids comprises increasing the number of identical nucleic acids in a sample, preferably by PCR methods known to the skilled artisan.
In the following invention components thereof are described in non-limiting examples
The separation of PNA and a complex of dihydrofolate reductase (DHFR) with PNA bound thereto is performed using a covalently polyacrylamide-coated capillary with an internal diameter of 75 μm, an overall length of 70 cm and an effective length (injection site to detector) of 51 cm. The separation buffer contains 20 mmol/l disodium hydrogenphosphate adjusted to pH 7.3 and at least 0.2 mmol/l sodiumdodecylsulfate (SDS). The applied voltage is 430 V/cm (anode at detector side). The capillary is temperated at 28° C. by airflow. Samples are injected hydrodynamically for 5 s and detected by absorption at 200 nm.
Using 0.6 mmol/l SDS DHFR could be detected after 8.8 min, using 0.2 mmol/l DHFR could be detected after 9.9 min. Higher concentrations of SDS (but below critical micellar concentration) had no significant effect on the migration speed. PNA could not be detected at all using this separation conditions.
Separation of PNA and DHFR/PNA complexes by HPLC is performed by using C18 reverse phase matrix in a column with an internal diameter of 1 mm and a length of 10 cm (particle diameter=2 μm). The mobile phases are 0.1 mol/l triethylammoniumacetate (pH 7) and acetonitrile at a flow rate of 40 μl/min. Substances are detected by absorption at 254 nm and 280 nm. The injected volume is 5 μl. Separation is started isocratically with 10% acetonitrile. Under these separation conditions protein fractions are detected in a time period of 2 to 6 min after sample injection. However the free PNA remains on the column and can be eluted and discarded together with all sample components by increasing the acetonitrile concentration in the mobile phase within a single step to 90%.
The synthesis of a 18-mer PNA with the sequence CCG ATT AAC GCT TGT ACC C is carried out using the synthesis processes described in the reference Thomson, S. A. et al., Fmoc mediated synthesis of peptide nucleic acids, Tetrahedron 1995, 51, 6179-94. In the same manner, different PNA 18-mers, differing in the nucleotide base sequence only, are synthesized in the same manner. The different PNA 18-mers are then joined in a solution forming the PNA library.
A solution comprising the PNA library according to the example 3 is contacted with DHFR under conditions preferably similar to those present in later applications for the selected PNA molecules and which are predominantly similar to that used in common SELEX experiments that are described in the references Tuerk, C., Gold, L., Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA Polymerase, Science 1990, 249(4968), 505-10 and Ellington, A. D. and Szostak, J. W., In vitro selection of RNA molecules that bind specific ligands. Nature 1990, 346(6287), 818-22. Then the solution is subjected to capillary electrophoresis or HPLC as a separation process according to example 1 or 2. Thereafter the isolated complexes are dissociated into DHFR and PNA either by incubation at high temperature, by pH-shift, competitive (by addition of existing ligands) or alternatively by degradation of DHFR using proteases or proteinases (e.g. Proteinase K).
Isolation of complementary ssDNA from an oligonucleotide library was performed by hybridization of matching oligonucleotides to a biotinylated 18mer PNA. 12 nmol of the oligonucleotide library (5′GAA TTC CAG ATC TCT NNN NNN NNN NNN NNN NNN GAT ATC AGG ATC CCA3′) was dissolved in 120 μl of buffer (10 mmol/l disodium hydrogenphosphate, pH 7.5). Different amounts of PNA (at least 105 molecules) were added to the reaction solution. The mixture was heated to 95° C. and the tubes were placed into a thermal isolated container (dewar) filled with hot water (92° C.). The container was isolated additionally with styrofoam and was cooled to room temperature over a time period of approximately five days. Generated PNA/DNA-hybrids were subsequently isolated by different methods and the DNA was amplified.
Isolation by PNA/DNA-Hybrids by immobilization on a solid phase and subsequent washing: 20 μl of an agarose matrix suspension with immobilized streptavidin was added to the hybridization solution containing the oligonucleotide library and PNA/DNA-hybrids. The mixture was shaked for 6 to 15 h and the suspension was transferred to a micro column (or alternatively into a filter pipet tip). The non hybridized DNA was removed by a flow of at least 100 column volumes of washing buffer (0.3 mol/l NaCl, 60 mmol/l Tris-HCl (pH 8.0), 2 mmol/l EDTA, 1% (w/v) SDS) through the column. Afterwards the matrix was washed with 1 ml 10 mmol/l Tris-HCl (pH 8.5). The PNA/DNA-hybrids were cleaved from the matrix specifically by reducing the disulfide bond in the biotinylation linker on the PNA with 10 mmol/l Tris-HCl (pH 8.5), 20 mmol/l DTT for 2 h at room temperature. The solution can be prepared for amplification of DNA by gel filtration (Sephadex G-25).
Isolation by PNA/DNA-Hybrids by immobilization on a solid phase and subsequent removal of free ssDNA in an electric field: 20 μl of an agarose matrix suspension with immobilized streptavidin (streptavidin coated magnetic beads can be used alternatively) was added to the hybridization solution containing the oligonucleotide library and PNA/DNA-hybrids. The mixture was shaked for 6 to 15 h and the suspension was transferred to a buffer (TBE) filled tube with 1% solid agarose in the bottom part. The agarose matrix with bound PNA/DNA-hybrids is transferred into the tube to settle on the agarose. The tube with an internal diameter of 5 mm was fixed between two buffer reservoirs and an electric field with a voltage difference of 100 V was applied over the tube for 1 to 5 hours leading to a migration of the free DNA into the agarose and subsequently into the anode buffer reservoir. Afterwards the matrix was washed with 1 ml 10 mmol/l Tris-HCl (pH 8.5). The PNA/DNA-hybrids were cleaved from the matrix specifically by reducing the disulfide bond in the biotinylation linker on the PNA with 10 mmol/l Tris-HCl (pH 8.5), 20 mmol/l DTT for 2 h at room temperature. The solution can be prepared for amplification of the DNA by gel filtration (Sephadex G-25). This separation method can also be applied in addition to preceding washing procedures.
Specific enzymatic degradation of non hybridized ssDNA with 51 nuclease: The specific degradation of non hybridized ssDNA with S1 nuclease was performed in 10 mmol/l Tris-acetic acid (pH 8.3), 50 mmol/l potassium acetate, 5 mmol/l magnesium acetate, 1 μg/ml BSA, 0.01% (v/v) Tween 20. Samples contained PNA (N-CCG ATT AAC GCT TGC ACC-C) and the oligonucleotides Pos2(3)D2-1rev (5′ATT TAT GAG GAG TCC GGT GCA AGC GTT AAT CGG GAT ATC AGG ATC CCA3′), Pos3(3)rev (5′GGA CTC CTC ATA AAT3′), Pos4 (5′TGG GAT CCT GAT ATC3′) in concentrations of 1 μmol/l each. The reactions were incubated for 2 min at 90° C. and afterwards slowly cooled to room temperature. After addition of S1 nuclease (50 or 100 Units) samples were incubated 3 h at 20° C.
Specific enzymatic degradation of non hybridized ssDNA with Mungobean nuclease: The specific degradation of non hybridized ssDNA with Mungobean nuclease was performed in 10 mmol/l Tris-acetic acid (pH 8.3), 50 mmol/l potassium acetate, 5 mmol/l magnesium acetate, 1 μg/ml BSA, 0.01% (v/v) Tween 20 (Zn2+ present in enzyme storage buffer). Samples contained PNA (N-CCG ATT AAC GCT TGC ACC-C) and the oligonucleotides Pos2(5)D2-1rev (5′ATT CTA TCA CGA GTC GGT GCA AGC GTT AAT CGG GAT ATG AGG ATC CCA3′), Pos3(5)rev (5′GAC TCG TGA TAG AAT3′), Pos4(5) (5′TGG GAT CCT CAT ATC3′) in concentrations of 1 μmol/l each. The reactions were incubated for 2 min at 90° C. and slowly cooled to room temperature. After addition of Mungo bean nuclease (20 or 10 Units) samples were incubated 15 h at 20° C.
Specific enzymatic degradation of non hybridized ssDNA with Type II restriction enzymes: The ssDNA contains a restriction site for a restriction enzyme Type II in the primer regions (
Specific enzymatic degradation of non hybridized ssDNA with Type IIs restriction enzymes: The sequence of ssDNA contains a recognition site for a Type IIs restriction enzyme in the primer regions of the ssDNA library (
Specific enzymatic degradation of non hybridized ssDNA with non-sequence specific nucleases (e.g. DNaseI): Since a PNA/DNA-Hybrid is not a substrate for the most nucleases, the degradation of non hybridized ssDNA can be performed by incubation with DNases after protection of necessary primer regions with PNA. If there is a matching PNA (
Separation of ssDNA and PNA/DNA-Hybrids with HPLC: HPLC separation of ssDNA and PNA/DNA-Hybrids was performed by using C4 reverse phase matrix in a column with an internal diameter of 4.6 mm and a length of 25 cm (particle diameter=5 μm). The mobile phases were 0.1 mol/l triethylammonium-acetate (pH 7) and acetonitrile at a flow rate of 1.3 ml/min. Substances were detected by absorption at 254 nm. Injection volume was 20 μl. Separation were executed with a gradient from 10 to 15% acetonitrile over 20 min.
Buffer system with wax separation for degradation and subsequent amplification of ssDNA in a closed system: Two buffer compartments with a volume of 25 μl each were generated in a 200 μl reaction tube by separation with a wax layer. This provides the possibility of mixing the buffers automatically by rising the temperature and without intervention into the system by rising the temperature (
Immobilized nucleases for degradation of ssDNA and subsequent removal of enzymes: Using immobilized nucleases to degrade ssDNA specifically will ease the subsequent removal of the nucleases prior to PCR amplification.
Chemical synthesis of PNA complementary DNA by using cyanogen bromide: For the chemical ligation of complementary DNA on a PNA template two 24 nucleotide long ssDNA were used that hybridized with nine nucleotides (
Chemical synthesis of PNA complementary DNA by using EDC: For the chemical synthesis of DNA on a PNA template two 24 nucleotide long ssDNA were used that hybridized with nine nucleotides each on an 18mer PNA (
Template-directed coupling of PNA-hybridized ssDNA-fragments with ligases: Synthesis of PNA-complementary DNA by coupling ssDNA-fragments in a template-directed manner on a PNA can be realized by closing the nicks between hybridized fragments using ligases. These ligases could be single- or doublestranded (for example but not restricted to: DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, DNA ligase V, T4 DNA ligase, Tag DNA ligase, T4 RNA ligase, T4 RNA ligase II, ThermoPhage™ single-stranded DNA ligase, Rma DNA ligase, Tsc DNA ligase, E. coli DNA ligase, LdMNPV DNA ligase, LigTK, Mth ligase, PBCV-1 DNA ligase, Pfu DNA ligase, Sealase, T4 ATP ligase, Vaccinia ligase, Tfi DNA ligase, Tth DNA ligase, Band IV, DREL, gp24.1, P52, RM378 RNA ligase, TbMP52, Rcl1p, DNA ligase D, XRCC4-ligase, T7 DNA ligase, Bst ligase, DraRnl)
Enzymatical ligation of PNA complementary DNA by using T4 RNA Ligase and ssDNA with an overhang: The oligonucleotides APos2(5)D2-1rev (5′ATT CTA TCA CGA GTC GGT GCA AGC3′) and pBPos2(5)D2-1rev (5′Pho-GTT AAT CGG GAT ATG AGG ATC CCA3′) hybridized with nine nucleotides each on a template molecule that consisted of PNA (N-CCG ATT AAC GCT TGC ACC-C) and were ligated enzymatically with 500 U/ml T4 RNA ligase in 50 mmol/l HEPES/NaOH (pH 8), 10 mmol/l magnesium chloride, 100 μmol/l ATP and 10 μg/ml BSA. Concentrations for the oligonucleotides and the PNA were 1 μmol/l. All components except T4 RNA ligase and BSA were heated to 94° C. for 2 min and cooled slowly to room temperature. Ligation reactions were started by addition of BSA and T4 RNA ligase prior to an incubation of 15 h at room temperature. Ligation products were analysed by a denaturating PAGE (15% polyacrylamide; 29:1, acrylamide:bisacrylamide) which is shown in
Enzymatical template-directed ligation of hexamer- and pentamer-ssDNA with T4 RNA ligase: Template-directed ligation of hexamer and pentamer ssDNA was realized in 50 mmol/l HEPES/NaOH (pH 8), 10 mmol/l magnesiumchloride, 100 μmol/l ATP and 10 μg/ml BSA with 330 U/ml T4 RNA ligase. The used oligonucleotides for hexamer ssDNA ligation were 6merA (5′Pho-GGT GCA3′), 6merB (5′Pho-AGC GTT3′), 6merC (5′Pho-AAT CGG3′) (
Enzymatical template-directed ligation of tetramer ssDNA into a gap with T4 RNA ligase: For ligation of tetramer ssDNA on a PNA template the oligonucleotides 4merLig5′ (5′CAT TAG TTG GTG CAA3′) and 4merLig3′ (5′Pho-TAA TCG GGA TCT GAG3′) (
Template-directed elongation of PNA-hybridized ssDNA with tetramer- and trimer-ssDNA: Elongation of the oligonucleotide APos2(5)(−3)D2-1rev (5′ATT CTA TCA CGA GTC GGT GCA3′) was performed using the PNA (N-CCG ATT AAC GCT TGC ACC-C) as template for ligation of 5′-phosphorylated 4mer-(4-mer-deg; 5′Pho-NNN N3′) or 3mer-ssDNA (3mer-deg; 5′Pho-NNN3′) fragments (
Template-directed synthesis of PNA-complementary DNA with ssDNA-fragments without overhang: Synthesis of PNA-complementary DNA can be performed by enzymatical or chemical coupling of ssDNA fragments with a length from nine to two nucleotides on a PNA template (
Restriction of directly ligated linker oligonucleotides by restriction endonucleases: Another method to prevent the amplification of directly joined linker oligonucleotides is their hydrolysis using nucleases (
Template-directed elongation of PNA-hybridized ssDNA with pyrophosphates: Synthesis of PNA-complementary DNA can be performed by elongation of an initiation nucleotide with a length of at least three nucleotides which hybridizes at the C-terminus of the PNA. Elongation can be executed by enzymatical and template-directed coupling of adenylated desoxyribonucleotides (A-5′ pp 5′-dN) or adenylated ribonucleotides (A-5′ pp5′-N) that represent an intermediary product in the process of ligation ligation according to the reference England, T. E. et al., Dinucleoside pyrophosphates are substrates for T4-induced RNA ligase, Proc. Nat. Acad. Sci. 1977, 74(11), 4839-42.
(1) A-5′pp5′-dN+dNnOH—>dNnpdN+AMP (catalyzed by ligase) or (2) A-5′pp5′-N+NnOH—>NnpN+AMP (catalyzed by ligase)
Template-directed elongation of PNA-hybridized ssDNA with 3′,5′-bisphosphatenucleosides: Synthesis of PNA-complementary DNA can be performed by elongation of an initiation oligonucleotide with a length of at least three monomers which hybridizes at the C-terminus of the PNA. Elongation can be executed by template-directed enzymatical coupling of 2′-deoxynucleoside-3′,5′-bisphosphates or nucleoside-3′,5′-bisphosphates (
Linear amplification of PNA-sequence information by ligase chain reaction: Usage of thermostable ligases offers the possibility of a linear amplification of complementary DNA on a PNA template. The synthesis of cDNA is executed by recurring changes of the temperature between a denaturating temperature to dissociate DNA from the PNA-template and an annealing and ligation temperature to hybridize and anneal ssDNA fragments on the template.
Using PNA/DNA-chimera with terminal DNA building hairpin loops for ligation and to provide primer regions: Using PNA/DNA chimeras with flanking DNA segments (
Using PNA/DNA-chimera with terminal DNA being elongated by terminal-nucleotidyl transferases to provide hybridization regions: Attached nucleotides (
Using chemically preactivated buildings blocks or monomers: Ligation of ssDNA fragments or monomers on a PNA template can be performed by using chemically preactivated building-blocks. These building blocks can be generated for instance by activation of a phosphate group at the 5′- or 3′-end of ssDNA by using carbodiimides (e.g. EDC) and imidazole or NHS (
After hybridization or generation of complementary ss nucleic acids according to example 5 the nucleic acids are sequenced according to the reference Sanger F. et al., DNA-sequencing with chain-terminating inhibitors, Proc. Nat. Acad. Sci. 1977, 74, 5463-5467 and Hunkapiller, T., Large-scale and automated DNA sequence determination, Science 1991, 254, 59-67. The sequence information obtained thereby is translated into complementary sequence information, which is the sequence information of the PNA. With this PNA sequence information PNA molecules with such sequence can be synthesized according to example 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05090287.3 | Oct 2005 | EP | regional |
