EP0478708A4 - Formation of triple helix complexes of double stranded dna using nucleoside oligomers - Google Patents
Formation of triple helix complexes of double stranded dna using nucleoside oligomersInfo
- Publication number
- EP0478708A4 EP0478708A4 EP19900917768 EP90917768A EP0478708A4 EP 0478708 A4 EP0478708 A4 EP 0478708A4 EP 19900917768 EP19900917768 EP 19900917768 EP 90917768 A EP90917768 A EP 90917768A EP 0478708 A4 EP0478708 A4 EP 0478708A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- oligomer
- guanine
- adenine
- dna
- nucleosidyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
- C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
Definitions
- the present invention is directed to novel methods of detecting and locating specific sequences in double stranded DNA using nucleoside oligomers which are capable of specifically complexing with a selected double stranded DNA structure to give a triple helix structure.
- Triple helical complexes containing cytosine and thymidine on the Hoogsteen (or third) strand have been found to be stable in acidic to neutral solutions, respectively, but have been found to dissociate on increasing pH. Incorporation of modified bases of T, such as 5-bromo-uracil and C, such as 5- methylcytosine, into the Hoogsteen strand has been found to increase stability of the triple helix over a higher pH range.
- cytosine (C) In order for cytosine (C) to participate in the Hoogsteen-type pairing, a hydrogen must be available on the 3-N of the pyrimidine ring for hydrogen bonding. Accordingly, in some circumstances, C may be protonated at N-3.
- DNA exhibits a wide range of polymorphic confor ⁇ mations, such conformations may be essential for biologi ⁇ cal processes. Modulation of signal transduction by sequence-specific protein-DNA binding and molecular interactions such as transcription, translation, and rep ⁇ lication, are believed to be dependent upon DNA conforma ⁇ tion.
- Nuclease-resistant nonionic oligodeoxynucleotides consisting of a methylphosphonate (MP) backbone have been studied in vitro and in vivo as potential anticancer, antiviral and antibacterial agents.
- ODN oligodeoxynucleotides
- MP methylphosphonate
- the 5*-3* linked internucleotide bonds of these analogs closely approximate the conformation of nucleic acid phosphodiester bonds.
- the phosphate backbone is rendered neutral by methyl substitution of one anionic phosphoryl oxygen; decreasing inter- and intrastrand repulsion due to the charged phosphate groups.
- Analogs with MP backbone can penetrate living cells and have been shown to inhibit mRNA translation in globin synthesis and vesicular stomatitis viral protein synthesis, and inhibit splicing of pre-mRNA in inhibition of HSV replication.
- Mechanisms of action for inhibition by the nonionic analogs include formation of stable complexes with complementary RNA and/or DNA.
- Nonionic oligonucleoside alkyl- and aryl-phosphonate analogs complementary to a selected single stranded foreign nucleic acid sequence can selectively inhibit the expression or function or expression of that particular nucleic acid without disturbing the function or expression of other nucleic acids present in the cell, by binding to or interfering with that nucleic acid.
- the use of complementary nuclease-resistant nonionic oligonucleoside methylphosphonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including Herpes simplex virus-1 is disclosed in U.S. Patent No. 4,757,055.
- anti-sense oligonucleotides or phosphoro- thioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral mRNA into protein has been proposed.
- the anti-sense constructs can bind to viral mRNA and were thought to obstruct the cells ribosomes from moving along the mRNA and thereby halting the translation of mRNA into protein, a process called "translation arrest” or "ribosomal-hybridization arrest.
- the inhibition of infection of cells by HTLV-III by administration of oligonucleotides complementary to highly conserved regions of the HTLV-III genome necessary for HTLV-III replication and/or expression is disclosed in U.S. Patent No. 4,806,463.
- the oligonucleotides were found to affect viral replication and/or gene expression as assayed by reverse transcriptase activity (replication) and production of viral proteins pl5 and p24 (gene expres ⁇ sion) .
- the present invention is also directed to novel modified Oligomers which are useful for preventing expression and/or functioning of a selected double stranded DNA sequence and which optionally include a DNA modifying group. Additionally, the present inven ⁇ tion is directed to novel Oligomers which comprise cyto- sine analogs.
- the present invention is also directed to formation of a triple helix structure by the interaction of a specific segment of double stranded DNA and an
- Oligomer which is sufficiently complementary to the DNA segment to read it and base pair (or hybridize) thereto.
- the present invention is directed to methods of detecting or recognizing a specific segment of double stranded DNA which comprises contacting said segment of DNA with an Oligomer which is sufficiently complementary to the sequence of purine bases in said segment of double stranded DNA or a portion thereof to hydrogen bond (or hybridize) therewith thereby giving a triple helix structure.
- the present invention is directed to methods of preventing or inhibiting expression or function of a specific segment of double stranded DNA having a given sequence which comprises contacting said DNA segment with an Oligomer sufficiently complementary to said double stranded DNA segment to form hydrogen bonds therewith, thereby giving a triple helix structure.
- the present invention is directed to methods wherein the DNA segment comprises a gene in a living cell and wherein formation of the triple helix structure permanently inhibits or inactivates said gene.
- said Oligomer is modified to incorporate a DNA modifying group which, after the Oligomer hydrogen bonds or hybridizes with the selected DNA sequence, is caused to react chemically with the DNA and irreversibly modify it.
- modifications may include cross-linking Oligomer and DNA by forming a covalent bond thereto, alkylating the DNA, cleaving said DNA at a specific location, or by degrading or destroying the DNA.
- the present invention provides novel nonionic alkyl- and aryl-phosphonate Oligomers which are sufficiently complementary to the purine sequence of a specific double stranded DNA segment to hydrogen bond and form a triple helix structure.
- Preferred are nonionic methylphosphonate Oligomers.
- One preferred class of methylphosphonate Oligomers comprises only purine bases.
- the present invention also provides Oligomers having nucleosidyl units in which a cytosine analog replaces cytosine and wherein said cytosine analog comprises a heterocycle which has a hydrogen available for hydrogen bonding at the ring position which corresponds to N-3 of cytosine and which is capable of forming two hydrogen bonds with a guanine base at neutral pH.
- Oligomers which comprise a modified sugar moiety having a lengthening link between the 5'-C and the 5*-OH and/or the 3'-C and 3'-OH are provided. These Oligomers comprising such modified sugar moieties may read purine bases on both strands of a segment of double stranded DNA.
- Oligonucleotides refers to nucleosidyl units which are connected by internucleosidyl phosphorus group linkages and includes oligonucleotides, nonionic oligo ⁇ nucleoside alkyl- and aryl-phosphonate analogs, phosphorothioate analogs of oligonucleotides, phosphoami- date analogs of oligonucleotides, neutral phosphate ester oligonucleotide analogs, and other oligonucleotide analogs and modified oligonucleotides.
- R is an alkyl or aryl group.
- Suitable alkyl or aryl groups include those which do not sterically hinder the phosphonate linkage or interact with each other.
- the phosphonate group may exist in either an "R” or an "S 11 configuration.
- Phosphonate groups may be used as inter- nucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
- Phosphodiester groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
- alkyl or aryl-phosphonate Oligomer refers to nucleotide Oligomers having internucleosidyl phosphorus group linkages wherein at least one alkyl or aryl phosphonate internucleosidyl linkage replaces a phosphodiester internucleosidyl linkage.
- methylphosphonate Oligomer refers to nucleotide Oligomers (or oligonucleo ⁇ tide analogs) having internucleosidyl phosphorus group linkages wherein at least one methylphosphonate inter- nucleosidyl linkage replaces a phosphodiester inter ⁇ nucleosidyl linkage.
- Thin Strand Oligomer refers to Oligomers which are capable of reading a segment of double stranded DNA and forming a triple helix structure therewith.
- nucleoside includes a nucleosidyl unit and is used interchangeably therewith and includes not only units having A, G, C, T and U as their bases, but also analogs and modified forms of the bases (such as 8- substituted purines) .
- the term "complementary" when referring to an Oligomer Third Strand refers to Oligomers having base sequences which hydrogen bond (and base pair or hybridize) with the purine base of a corresponding (Watson-Crick) base pair of a double stranded DNA to form a triple helix structure.
- p in, e.g., as in ApA represents a phosphodiester linkage
- E in, e.g., as in C ⁇ G represents a methyl ⁇ phosphonate linkage.
- notation such as "T” indicates nucleosidyl groups linked by methyl phosphonate linkages.
- purine or “purine base” includes not only the naturally occurring adenine and guanine bases, but also modifications of those bases such as bases substituted at the 8-posi ion.
- read refers to the ability of a nucleic acid to recognize through hydrogen bond interactions the base sequence of another nucleic acid.
- a Third Strand Oligomer is able to recognize through hydrogen bond interactions the base pairs, in particular the purine bases, in the duplex of a segment of double stranded DNA.
- triplet refers to a situation such as that depicted in Figures 1A, IB and 2A to 2D wherein a base in the Third Strand has hydrogen bonded (and thus base paired) with a (Watson-Crick) base pair of a segment of double stranded DNA.
- Fig. 1A and IB depict triplets wherein a pyri idine base in the Third Strand forms a triplet with the duplex DNA (Watson-Crick) base pair.
- Fig. 2A to 2D depict triplets wherein a purine base in the Third Strand forms a triplet with the duplex DNA (Watson-Crick) base pair.
- Fig. 3A to 3C depict the base sequences of exemplary polypurine sequence triple helix structures wherein the Third Strand "reads" and, thus, base pairs with purine bases on one strand of the double stranded DNA.
- Fig. 4A to 4E depict the base sequences of exemplary mixed sequence triple helix structures wherein the Third Strand "reads" and, thus base pairs with purine bases on both strands of the double stranded DNA.
- Fig. 5 depicts a nucleosidyl unit having a modified sugar moiety with an alkyleneoxy link for lengthening internucleoside phosphorus linkages and processes for its preparation.
- Fig. 6 depicts a nucleosidyl unit having a modified sugar moiety with an alkylene link for lengthening internucleoside phosphorus linkages and processes for its synthesis.
- Fig. 7 depicts CD spectra of triple helix structures
- ( ) depicts an MP-Oligomer Third Strand
- Figs. 8A and 8B depict cross-linking of (A) single stranded DNA and (B) double stranded DNA using psoralen ⁇ derivatized MP-Oligomers.
- the present invention involves the formation of triple helix structures with a selected double stranded DNA sequence by contacting said DNA with an Oligomer which is sufficiently complementary to the purine sequence of the double stranded DNA to form hydrogen bonds (or hybri ⁇ dize) therewith.
- this invention includes the following aspects.
- a first aspect concerns the reading (or recogni- tion) of the base pairs in the double stranded DNA segment without opening the base pair, through the hydrogen bond formation by the bases in the Third Strand with the extra hydrogen bond sites of purines in the double stranded DNA, such as adenine and guanine.
- the reading of the base pair sequence in the DNA duplex is always done by reading the purine of the base pair, through hydrogen bond formation with the bases in the Third Strand, with the remaining available hydrogen bonding sites of the purines in the DNA.
- Either purines (such as adenine (A) or guanine (G) ) or pyrimidines (thymine (T) or cytosine (C) ) in the Third Strand can form hydrogen bonds with the purines in the DNA, i.e.: i) Adenine (A) in the base pair of DNA can be read or hydrogen bonded with A or T in the third strand. ii) Guanine (G) in the base pair of DNA can be read or paired with C or G in the third strand.
- the phosphorus-containing backbone of the Third Strand comprises methylphosphonate groups as well as naturally occurring phosphodiester groups.
- a second aspect of this invention is to read the purine (A or G) in the double stranded DNA totally by the use of purine bases from the Third Strand.
- this aspect of the present invention uses only G to read G in the base pairs in the DNA duplex, and similarly to use only A to read A in the base pairs in the DNA duplex.
- the polarity of the strands, either the anti- parallel or parallel direction of the Third Strand in respect to the strand containing the purine to be read in the duplex is important.
- the base planes of the purines and pyrimidines are rigid, and the furanose ring only allows a small ripple (about 0.5A above or below the plane).
- the conformational state of the nucleoside is defined prin ⁇ cipally by the rotation of these two more or less rigid planes, i.e. the base and the pentose, relative to each other about the axis of the C'-l to N-9 or N-l bond.
- the sugar-base torsion angle, ⁇ CT has been defined as "the angle formed by the trace of the plane of the base with the projection of the C-l 1 to 0-1' bond of the furanose ring when viewed along the C'-l to a bond.
- This angle will be taken as zero when the furanose-ring oxygen is antiplanar to C-2 of the pyrimidine or purine ring and positive angles will be taken as those measured in a clockwise direction when viewing C-l 1 to N.”
- This angle has also been termed the glycosyl torsion angle. Using the above definition, it was concluded that there were two ranges of ⁇ for the nucleosides, about -30° for the anti conformation and about +150° for the svn conformation. The range for each conformation is about ⁇ 45°. (22, 22a) Other researchers have used or proposed slightly different definitions of this angle.
- the conformations of the purine nucleo- sidyl units in the Third Strand are influenced by the polarity (parallel (5' to 3') or anti-parallel (3 « to 5 « ) direction) of the strand containing the purine bases to be read in the DNA in relation to the Third Strand.
- the conforma ⁇ tion of the purine nucleosidyl unit in the Third Strand should be in the syn conformation.
- the conformation of the purine nucleosidyl unit in the Third Strand in reading the corresponding purine in the anti-parallel strand in the duplex should exist in anti conformation.
- a Third Strand which has the same polarity as (i.e. is parallel to) either one strand or the opposite strand.
- the Third Strand parallel to the Crick strand (the Third Strand parallel to the Crick strand)
- the purine nucleosidyl units (A or G) of the Third Strand need to be in syn conformation in reading the purines in the base pair (A or G) of the parallel ("Watson") strand of the double stranded DNA.
- the second purine in the second base pair the same requirement applies if the purine is located in the same strand as the first purine.
- the purine nucleo- sidyl unit needs to be in anti conformation.
- adenine in the Third Strand is used to read adenine in the duplex and guanine in the Third Strand is used to read guanine in the duplex.
- a third aspect of this invention concerns the length of the linkage of the phosphorus backbone of the Third Strand to allow reading of the purine bases on either strand of the double stranded DNA.
- the distance between nucleosidyl units along the phosphorus backbone must be increased.
- Two examples of types of lengthening link formats for the phosphorus backbone are proposed.
- One type of link format for the phosphorus backbone would use a universal lengthening link on the individual nucleosidyl units, i.e. all the length- ening links of the Third Strand would be the same.
- Such a universal link format is particularly suitable for Third Strands comprising only purine bases.
- the length of the link between the 5' carbon of the "nucleo ⁇ sidyl unit one" to the 3* oxygen of the subsequent "nucleosidyl unit two” may be increased by two atoms (such as -CH-CH--) or by 3 atoms (such as -0-CH 2 -CH 2 -) , thereby lengthening the linkage between individual nucleosidyl units by 2 to 6__.
- separation of the units be increased by a number of atoms ranging from 1 to 6.
- Figure 6 illustrates an example of a nucleosidyl unit comprising this lengthening link format and proposed synthetic routes.
- a second lengthening link format for the phosphorus backbone would comprise non-uniform lengthening links.
- Links having internucleosidyl distances on the order of the standard phosphodiester backbone for the Third Strand would be employed when the purines being read were on the same strand, while a lengthened link (15-17A in length) which could comprise lengthening links on the 3* carbon of one nucleosidyl unit and on the 5 1 - carbon of its neighbor, would be employed to read the purine bases located on opposite strands.
- Such a non-uniform lengthen ⁇ ing link format would be particularly suitable for use in Third Strands comprising both pyrimidine (or pyrimidine analog) and purine bases.
- triplets are formed wherein a pyrimidine base in the Third Strand forms hydrogen bonds and, thus, base pairs with the purine base of a base pair of the double stranded DNA sequence.
- Examples of such triplets where having a pyrimidine base as the base in the Third Strand are shown in Figures 1A and IB.
- Fig. 1A depicts a triplet having T as the Third Strand base which forms hydrogen bonds and, thus, base pairs with the A of the double stranded DNA.
- the T of the Third Strand is aligned parallel to the A-containing strand of the double stranded DNA, and anti-parallel to the T-containing strand of the double stranded DNA (which is also anti-parallel to the A- containing strand) . Accordingly, the sequence for that triplet is written as follows:
- Fig. IB depicts a triplet having a protonated cytosine (C+) as the Third Strand base which forms hydro- gen bonds and, thus, base pairs with a G of a G-C base pair in the double stranded DNA.
- the cytosine base in the Third Strand must be protonated at N3 in order to form hydrogen bonds necessary for a stable triplet.
- a cytosine may be replaced with a cytosine analog having a quaternary nitrogen at a position analogous to N3.
- the C+ (or its analog) of the Third Strand is aligned parallel to the G-containing strand of the double stranded DNA, and anti-parallel to the C-containing strand of the double stranded DNA (which is also anti-parallel to the G-con ⁇ taining strand) . Accordingly, such a triplet sequence is written as follows:
- triplets are formed wherein a purine base in the Third Strand forms hydrogen bonds with and, thus, base pairs with the same purine base in one strand of the double stranded DNA.
- A will pair with A and G will pair with G.
- the purine base in the Third Strand is capable of base pairing with the purine base of double stranded DNA such that the strand polarity of the Third Strand containing the purine base may be aligned either parallel or anti- parallel to strand polarity of the strand containing the purine to be read in the double stranded DNA.
- Fig. 2A depicts a triplet where the A of the Third Strand is aligned parallel to the A of the double stranded DNA, and anti-parallel to the T of the double stranded DNA.
- the glycosyl (C-N) torsion angle of the Third Strand A is in the svn conformation, and the glycosyl torsion angles of the A and T bases of the double stranded DNA are both in the anti conformation.
- Fig. 2B depicts a triplet where the A of the Third Strand is aligned anti-parallel to the A of the double stranded DNA and parallel to the T of the double stranded DNA.
- the glycosyl torsion angles of all three bases are in the anti confor ⁇ mation.
- Fig. 2C depicts a triplet wherein the G of the Third Strand is aligned parallel to the G of the double stranded DNA, and anti-parallel to the C of the double stranded DNA.
- the glycosyl torsion angle of the Third Strand G is in the syn conformation, and the glycosyl torsion angles of the G-C bases of the double stranded DNA are both in the anti conformation.
- Such a triplet sequence is written as follows:
- Fig. 2D depicts a triplet wherein the G of the Third Strand is aligned anti-parallel to the G of the double stranded DNA and parallel to the C of the double stranded DNA.
- the glycosyl torsion angles for all three bases are in the anti conformation.
- an Oligomer complementary to the polypurine sequence is used and may comprise a complementary sequence of pyrimidines or purines or a mixture thereof.
- Preferred Oligomers include nonionic MP-Oligomers which are nuclease resistant and are capable of forming the previously-discussed triplet structures with the double stranded DNA. Examples of triple stranded helix sequences wherein the Third Strand binds to a polypurine sequence in one strand of a double stranded DNA sequence are schemati ⁇ cally depicted in Fig. 3A to 3C. In Figures 3A to 3C, C + denotes a protonated C.
- Mixed sequences are sequences wherein the purine bases of the selected DNA sequence are located on both strands of the double stranded DNA sequence. Accordingly, the Third Strand Oligomer must be able to hydrogen bond and, thus, base pair with the purine base of each base pair of the double stranded DNA sequence without regard to which strand of the double stranded DNA the purine base is located on. Accordingly, the Third Strand Oligomer must be able to "read” across the double stranded DNA. Ex- amples of mixed sequences including an appropriately complementary Third Strand are depicted in Fig. 4A to 4E. Fig. 4A depicts a mixed sequence having a pyrimidine- rich Third Strand.
- Fig. 4B depicts a mixed sequence having a purine- rich Third Strand.
- Fig. 4C, 4D and 4E depict mixed sequences having Third Strands containing only purine bases.
- S denotes syn and "a” denotes anti: no super ⁇ script denotes anti.
- no super ⁇ script denotes anti.
- an internucleosidyl linkage may be lengthened by the interposition of an appropriate alkylene (-(CH 2 ) n -) or alkyleneoxy (-(CH 2 ) n O-) lengthening link between the 5*-carbon and the 5* hydroxyl of the sugar moiety of a nucleosidyl unit or a similar link between the 3'-carbon and the 3'-hydroxyl. (See Figures 5 and 6). Where indicated, such lengthening links may be interposed at both the 3'- and 5'- carbons of the sugar moiety.
- internucleosidyl phosphorus linkages such as methylphosphonate linkages allow an appropriate base on the Third Strand to read consecutive purine bases on one strand of the selected double stranded DNA sequence.
- Oligomers which are capable of reading with the purine bases on both strands of a selected double stranded DNA sequence, may be prepared.
- Oligomers having at least about 7 nucleosides which is usually a sufficient number to allow for specific binding to a desired purine sequence of a segment of double stranded DNA. More preferred are Oligomers having from about 8 to about 40 nucleotides; especially preferred are Oligomers having from about 10 to about 25 nucleosides. Due to a combination of ease of synthesis, with specificity for a selected sequence, coupled with minimization of intra-Oligomer, internucleo ⁇ side interactions such as folding and coiling, it is believed that Oligomers having from about 14 to about 18 nucleosides comprise a particularly preferred group.
- Oligomers may comprise either ribosyl moieties or deoxyribosyl moieties or modifications thereof.
- Oligomers comprising deoxyribosyl or modified deoxyribosyl moieties are preferred.
- nucleotide Oligomers i.e., having the phosphodiester internucleoside linkages present in natural nucleotide Oligomers, as well as other oligonucleotide analogs
- preferred Oligomers comprise oligonucleoside alkyl and arylphosphonate analogs, phosphorothioate oligonucleoside analogs, phosphoroamidate analogs and neutral phosphate ester oligonucleotide analogs.
- oligonucleoside alkyl- and aryl-phosphonate analogs in which phosphonate linkages replace one or more of the phosphodiester linkages which connect two nucleo- sidyl units.
- MP-Oligomers Preferred synthetic methods for methylphosphate Oligomers are described in Lee, B.L. ejt al. Biochemistrv 27:3197-3203 (1988) and Miller, P.S., e£ al.. Biochemistrv 25_:5092-5097 (1986), the disclosures of which are incorporated herein by reference.
- oligonucleosidyl alkyl- and aryl ⁇ phosphonate analogs wherein at least one of the phospho- diester internucleoside linkages is replaced by a 3* - 5' linked internucleoside methylphosphonyl (MP) group (or "methylphosphonate”) .
- MP internucleoside methylphosphonyl
- the methylphosphonate linkage is isosteric with respect to the phosphate groups of oligo ⁇ nucleotides.
- MP-oligomers should present minimal steric restric- tio.ns to interaction with the selected DNA sequences.
- MP-Oligomers should be more resistant to hydrolysis by various nuclease and esterase activities, since the methylphosphonyl group is not found in naturally occurring nucleic acid molecules. Due to the nonionic nature of the methylphosphonate linkage, these MP-oligomers should be better able to cross cell membranes and thus be taken up by cells.
- MP-Oligomers are more resistant to nuclease hydrolysis, are taken up in intact form by mammalian cells in culture and can exert specific inhibitory effects on cellular DNA and protein synthesis (See, e.g., U.S. Patent No, 4,469,863).
- MP-Oligomers having at least about seven nucleosidyl units, more preferably at least about 8, which is usually sufficient to allow for specific recogni ⁇ tion of the desired segment of double stranded DNA. More preferred are MP-Oligomers having from about 8 to about 40 nucleosides, especially preferred are those having from about 10 to about 25 nucleosides.
- MP-Oligomers of from about 14 to 18 nucleosides.
- MP-Oligomers where the 5'-internucleosidyl linkage is a phosphodiester linkage and the remainder of the internucleosidyl linkages are methylphosphonyl linkages. Having a phosphodiester linkage on the 5' - end of the MP-Oligo er permits kinase labelling and electrophoresis of the Oligomer and also improves its solubility.
- the selected double stranded DNA sequences are sequenced and MP-Oligomers complementary to the purine sequence are prepared by the methods disclosed in the above noted patents and disclosed herein.
- Oligomers are useful in determining the presence or absence of a selected double stranded DNA sequence in a mixture of nucleic acids or in samples including isolated cells, tissue samples or bodily fluids.
- Oligomers are useful as hybridization assay probes and may be used in detection assays. When used as probes, these Oligomers may also be used in diagnostic kits. If desired, labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercalat ⁇ ing agents such as acridine, or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular scissors like o-phenanthrolinecopper or ED- TA-iron may be incorporated in the MP-Oligomers.
- labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercalat ⁇ ing agents such as acridine, or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular scissors like o-phenanthrolinecopper or ED- TA-iron may be incorporated in the MP-Oligomers.
- Oligomers may be labelled by any of several well known methods.
- Useful labels include radioisotopes as well as nonradioactive reporting groups.
- Isotopic labels include 3 H, 35 S, 32 P, 125 I, Cobalt and U C.
- Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcription.
- hybridi ⁇ zation can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radioisotope used for labelling.
- Non-isotopic materials can also be used for labelling, and may be introduced by the incorporation of modified nucleosides or nucleoside analogs through the use of enzymes or by chemical modification of the Oligomer, for example, by the use of non-nucleotide linker groups.
- Non-isotopic labels include fluorescent molecules, chemi- luminescent molecules, enzymes, cofactors, enzyme sub ⁇ strates, haptens or other ligands.
- One preferred labell ⁇ ing method includes incorporation of acridinium esters. Such labelled Oligomers are particularly suited as hybridization assay probes and for use in hybridization assays.
- these Oligomers When used to prevent function or expression of a double stranded DNA sequence, these Oligomers may be advantageously derivatized or* modified to incorporate a DNA modifying group which may be caused to react with said DNA and irreversibly modify its structure, thereby render ⁇ ing it non-functional.
- a DNA modifying group which may be caused to react with said DNA and irreversibly modify its structure, thereby render ⁇ ing it non-functional.
- Our co-pending patent application U.S. Serial No. 924,234, filed October 28, 1986, the disclosure of which is incorporated herein by reference, teaches the derivatization of Oligomers which comprise oligonucleoside alkyl and arylphosphonates and the use of such derivatized oligonucleoside alkyl and arylphosphonates to render targeted single stranded nucleic acid sequences non-functional.
- DNA modifying groups may be used to derivatize these Oligomers.
- DNA modifying groups include groups which, after the derivatized Oligomer forms a triple helix structure with the double stranded DNA segment, may be caused to form a covalent linkage, cross ⁇ link, alkylate, cleave, degrade, or otherwise inactivate or destroy the DNA segment or a target sequence portion thereof, and thereby irreversibly inhibit the function and/or expression of that DNA segment.
- the location of the DNA modifying groups in the Oligomer may be varied and may depend on the particular DNA modifying group employed and the targeted double stranded DNA segment. Accordingly, the DNA modifying group may be positioned at the end of the Oligomer or intermediate the ends.
- a plurality of DNA modifying groups may be included.
- the DNA modifying group is photoreactable (e.g., activated by a particular wave ⁇ length, or range of wavelengths of light) , so as to cause reaction and, thus, cross-linking between the Oligomer and the double stranded DNA.
- DNA modifying groups which may cause cross-linking are the psoralens, such as an amino- methyltrimethyl psoralen group (AMT) .
- AMT amino- methyltrimethyl psoralen group
- Other cross-linking groups which may or may not be photoreactable may be used to derivatize these Oligomers.
- the DNA modifying groups may comprise an alkylating agent group which, on reaction, separates from the Oligomer and is covalently bonded to the DNA segment to render it inactive.
- alkylating agent groups are known in the chemical arts and include groups derived from alkyl halides, haloacetamides, phosphotriesters and the like.
- DNA modifying groups which may be caused to cleave the DNA segment include transition metal chelating complexes such as ethylene diamine tetraacetate (EDTA) or a derivative thereof.
- transition metal chelating complexes such as ethylene diamine tetraacetate (EDTA) or a derivative thereof.
- Other groups which may be used to effect cleaving include phenanthroline, porphyrin or bleo ycin, and the like.
- iron may be advantageously tethered to the Oligomer to help generate the cleaving radicals.
- EDTA is a preferred DNA cleaving group
- other nitrogen containing materials such as azo compounds or nitreens or other transition metal chelating complexes may be used.
- the nucleosidyl units of Third Strand Oligomers which read purine bases on both strands of a double stranded DNA sequence may comprise a mixture of purine and pyrimidine bases or only purine bases.
- purine bases on both strands of a double stranded DNA sequence are to be read, it is preferred to use Oligomers having only purine bases. It is believed that such purine-only Oligomers are advantageous for several reasons: (a) purines have higher stacking proper ⁇ ties than pyrimidines, which would tend to increase stability of the triple helix structure; (b) use of purines only eliminates the need for either protonation of cytosine (so it has an available hydrogen for hydrogen bonding at the N-3 position at neutral pH) or use of a cytosine analog having such an available hydrogen at the position which corresponds to N3 on the pyrimidine ring; and allows use of a universal lengthening link.
- the purine bases are normally in the anti conformation; however, the barrier for a base to roll over to the svn conformation is low.
- the purines on the Third Strand may assume the syn conformation during the hydrogen bonding process.
- the purine may be modified at the 8-position with a substitutent such as methyl, bromo, isopropyl or other bulky group so it will assume the syn configuration under normal conditions. Nucleosidyl units comprising such substituted purines would thus normally assume the syn conformation.
- the present invention contemplates the optional incorporation of such 8-substituted purines in place of unsubstituted A or G. Studies with our (Kendrew) models indicate that such substitutions should not affect formation of the triple helix structure.
- purine nucleosidyl units in the anti and syn conformations, as appropriate (following the rules for reading the double stranded DNA described herein) allows reading of the purines on both strands of the duplex and formation of a triple helix structure by the purine-only third strand with the double stranded DNA. If a Third Strand Oligomer comprising both pyrimides and purines is used to read purines on both strands of a double stranded DNA, a non-uniform link format is used as described herein to allow the third strand to read across from one strand of the duplex to the other.
- the present invention provides a novel class of purine Oligomers which comprise nucleosidyl units selected from:
- Bp is a purine base
- R is independently selected from alkyl and aryl groups which do not sterically hinder the phosphonate linkage or interact with each other
- R' is hydrogen, hydroxy or methoxy
- alk is alkylene of 2 to
- Oligomers which comprise at least about
- nucleosidyl units where R is methyl. Also preferred are nucleosidyl units wherein R* is hydrogen.
- Suitable bases Bp include adenine and guanine, either optionally substituted at the 8-position, preferred substitutions include methyl, bromo, isopropyl and the like.
- novel Oligomers comprise nucleosidyl units wherein cytosine has been replaced by a cytosine analog comprising a heterocycle. which has an available hydrogen at the ring position analogous to the 3-N of the cytosine ring and is capable of forming two hydrogen bonds with a guanine base at neutral pH and thus does not require protonation as does cytosine for Hoogstein-type base pairing, or formation of a triplet.
- Suitable nucleosidyl units comprise analogs having a six-membered heterocyclic ring which has a hydrogen available for hydrogen bonding at the ring position corresponding to N-3 of cytosine and which is capable of forming two hydrogen bonds with a guanine base at neutral pH and include 2'-deoxy-5,6-dihydro-5-azadeoxycytidine (I), pseudoisocytidine (II), 6-amino-3-(0-D-ribofuran- osyl)pyrimidine-2,4-dione (III) and l-amino-l,2,4-( / 9-D- deoxyribofuranosyl)triazine-3-[4H]-one (IV), the structures of which are set forth in Table 5.
- Oligomers comprising nucleosidyl units which comprise modified sugar moieties having lengthening links may be convenient ⁇ ly prepared by these methods. Oligomers comprising phosphodiester internucleosidyl phosphorus linkages may be synthesized using any of several conventional methods, including automated solid phase chemical synthesis using cyanoethylphosphoroamidite precursors (29) . If desired, the previously-described nucleosidyl units comprising cytosine analogs (see Table 5) may be incorporated into the MP-Oligomer by substituting the appropriate cytidine analog (see Table 5) in the reaction mixture.
- MP-Oligomers may be prepared using modified nucleosides where either the bond between the 5'-carbon and the 5'-hydroxyl or the 3'-carbon and the 3'-hydroxyl of the sugar moiety has been substituted with a alkyle- neoxy group, such as ethyleneoxy group.
- Figure 5 shows proposed reaction schemes for preparation of intermediates for modified nucleosides having either a 3'-(ethyleneoxy) or 5'-(ethyleneoxy) link.
- B represents a base
- Tr and R represent protecting groups
- nucleosidyl units having such lengthening links at both the 3'- and 5'- positions of the sugar moiety may be prepared.
- MP-Oligomers having a slightly lengthened internucleoside link on the phosphorus backbone.
- Such MP-Oligomers may be prepared using nucleosides in which the sugar (deoxyribosyl or ribosyl) moiety has been modified to replace the 5'-hydroxy with a ⁇ - hydroxyethyl (HO-CH 2 -CH--) group synthetic schemes for the preparation of such a 5*-0-hydroxyethyl-substituted nucleosides is depicted in Figure 6.
- Figure 6 depicts a proposed reaction scheme for a 5'- ⁇ -hydroxyethyl-substituted sugar analog.
- DCC denotes dicyclohexylcarbodiimide
- DMSO denotes dimethylsulfoxide.
- B is a base.
- Suitable protecting groups, R include t-butyldimethyl silyl and tetrahydro- pyranyl.
- MP-Oligomers incorporating the above-described modified nucleosidyl units are prepared as described above, substituting the modified nucleosidyl unit.
- Oligomers comprising only purine bases
- nucleosidyl units having the same lenthening links may be employed.
- a mixture of nucleosidyl units having no lengthening link and lengthen ⁇ ing links are used; nucleosidyl units having lengthening links at both the 3'- carbon and the 5'-carbon of the sugar moiety may be advantageous.
- Derivatized Oligomers may be readily prepared by adding the desired DNA modifying groups to the Oligomer.
- the number of nucleosidyl units in the Oligomer and the position of the DNA modifying group(s) in the Oligomer may be varied.
- the DNA modifying group(s) may be positioned in the Oligomer where it will most effectively modify the target sequence of the DNA. Accordingly, the positioning of the DNA modifying group may depend, in large measure, on the DNA segment involved and its key target site or sites, although such optimum position can be readily determined by conventional techniques known to those skilled in the art.
- MP-Oligomers such as 8-methoxypsoralen and 4'-aminomethyl- trimethylpsoralen (AMT)
- AMT 4'-aminomethyl- trimethylpsoralen
- a specific segment of double stranded DNA may be detected or recog ⁇ nized using an MP-Oligomer Third Strand which reads the purine bases of the duplex of the double-stranded DNA according to the triplet base pairing guidelines described herein.
- the MP-Oligomer Third Strand has a sequence selected such that the base of each nucleosidyl unit will form a triplet with a corresponding base pair of the duplex DNA to give a triple helix structure.
- Detectably labeled Oligomers may be used as probes for use in hybrid ⁇ ization assays, for example, to detect the presence of a particular double-stranded DNA sequence.
- the present invention also provides a method of preventing expression or function of a selected sequence in double stranded DNA by use of an MP-Oligomer which reads the DNA sequence and forms a triple helix structure. Formation of the triple helix may prevent expression and/or function by modes such as preventing opening of the duplex for transcription, preventing of binding of effec- tor molecules (such as proteins) , etc.
- Derivatized Oligomers may be used to detect or locate and then ir ⁇ reversibly modify at target site in the DNA duplex by cross-linking (psoralens) or cleaving one or both strands (EDTA) . By careful selection of a target site for cleavage, the Oligomer may be used as a molecular scissors to specifically excise a selected DNA sequence.
- the Oligomers may be derivatized to incorporate a DNA reacting or modifying group which can be caused to react with the DNA segment or a target sequence thereof to irreversibly modify, degrade or destroy the DNA and thus irreversibly inhibit its functions.
- these Oligomers may be used to irreversibly inactivate or inhibit a particular gene or target sequence of the game in a Irving' cell, allowing selective inactivation or inhibition. These Oligomers could then be used to permanently inactivate, turn off or destroy genes which produced defective or undesired products or if activated caused undesirable effects.
- Another aspect of the present invention is directed to a kit for detecting a particular double stranded DNA sequence which comprises a detectably labeled purine MP- Oligomer Third Strand selected to be able sufficiently complementary to the purine sequence to be able to read the sequence and form a triple helix structure.
- Triple-strandedpoly(dT 10 )-poly(dA 10 )-poly(dT 10 ) [T ⁇ T g ] coordinates were obtained from the A-DNA x-ray structure of Arnott and Seising (10) . The same coordinates were used for the starting geometry of poly(dT 10 )-poly (dA 10 )-poly(dT 10 ) methylphosphonate [T 1 AT 2 MP] . Geometry optimization and partial atomic charge assignments for the dimethyl ester methylphosphonate fragment were calculated by afe j-nitio quantum mechanical methods with QUEST (version 1.1) using 3-21G* and STOG* basis sets, respectively (11) .
- the latter basis set was used to maintain uniform charge assignments with those previously calculated for nucleic acids in the AMBER database.
- the final monopole atomic charge assignments for the MP fragment were made to obtain a neutral net charge for each base, furanose, and MP backbone of the third DNA strand.
- Alternating R p . and S p _ methyl substitution of the backbone phosphoryl oxygens of the T 2 MP strand was done by stereo computer graphics.
- the substitution of MP diastereomers was made in this manner to approximate experimental yield, since the synthesis cannot be controlled.
- Molecular mechanics and molecular dynamics calculations were made with a fully vectorized version of AMBER (version 3.1), using an all-atom force field (12, 13). All calculations were performed on CRAY X-MP/24 and VAX 8600 computers.
- the negative charge of the DNA phosphate backbone was rendered neutral by placement of positive counterions within 4 A of the phosphorus atoms bisecting the phosphate oxygens; counterions were not placed on the MP-substituted strand.
- the triple helices and counterions were surrounded by a 10A shell of TIP3P water (14) molecules with periodic boundary conditions.
- the box dimensions were 101, 686.8 A 3 for T ⁇ T-, and 124,321.1 A 3 for T ⁇ T ⁇ .
- the third DNA strand with MP backbone resulted in several changes consistent with enhanced binding of the ODN with the MP backbone in the triple helix.
- the average hydrogen bond distances and mean atomic fluctuations are consistently smaller in the T ⁇ T-MP triplet (Table 1)
- the interstrand phosphorus atoms distance was 9.6A (+/-0.91) for A-T 2 and 8.3A (+/-0.58) for A-T-MP.
- the reduced interstrand phosphorus atom distance and smaller mean atomic fluctuations between the second and third strands are due to decreased interstrand electrostatic repulsion accompanying MP substitution in the backbone.
- the sugar puckering pattern of the MP substituted helix had a greater proportion of 01'endo and C2'endo conformations in contrast to the unsubstituted helix.
- Analysis of other conformational parameters support the hybrid conformational nature of these triple helices.
- the helical twist angle (between Tl and A strands) averaged 39.4 degrees (+ ⁇ -2.86) for the T ⁇ T j structure and is more consistent with a B-DNA conformation (range 36-45) .
- the T ⁇ T j MP helical twist angle averages 32.0 degrees (+ ⁇ -2.19) and is closer to that of A-DNA (range 30-32.7).
- the average helical repeat singles (between the Tl and A strands) for the entire structure are for 10.2 TAT 2 and 11.2 degrees for T ⁇ T-MP.
- the average intrastrand phosphorus atom distances over the 40 psec trajectory are presented in Table 3.
- the intrastrand phosphorus distances of the T 1 strands are most consistent with an A-DNA conformation (7.0 A).
- the interstrand phosphorus distances of the T ⁇ strand are more consistent with a B-DNA conformation, in contrast to values more consistent with A-DNA for the T 2 strand.
- Comparisons of the ⁇ -dihedral of the adenine strands reveals a slight change in the average position of the dihedral with MP substitution, positioning the dihedral closer to trans, but there is a large overlap in the transitional motions of both dihedrals during the 40 psec trajectory. There was a significant change (by 27.0 degrees) in the average Sp-MP diastereomer ⁇ dihedral angle from baseline. The fluctuation of the ⁇ dihedral containing the Sp-MP diastereomer was significantly less than the Rp-MP.
- the conformation of these DNA structures differs from experimental data based on the fiber diagram.
- the structure of poly(dT)poly(dA)-poly(dT) was determined by x-ray diffraction studies under conditions of 92% humidity, and is a low resolution structure (10) .
- the molecular dynamics simulations are of fully solvated DNA structures under periodic boundary conditions with counterions. DNA in solution is generally believed to predominate in the B-form; A-DNA conformation predominates under conditions of lower humidity (16) .
- Several triple-stranded DNA helical structures have been determined by x-ray diffraction studies and have been uniformly observed in an A-DNA conformation under conditions of low humidity and increased salt concentration (10,17,18).
- Circular dichroism spectroscopy studies were performed using Triple Helix Structures formed using a combination following nucleoside oligomers.
- Psoralen derivatized dTp(l) 6 oligomers were prepared as described in Lee, B.L., ⁇ _ &_.. , Biochemistry 27:3197- 3203 (1988).
- the T 7 oligomers were allowed to hybridize with DNA having the following sequence including a 15-mer poly A subsequence:
- Figure 8A shows crosslinking of the psoralen derivatized T 7 Oligomers with the single stranded (poly A containing) DNA sequence.
- Figure 8B shows crosslinking of the double stranded DNA with the double stranded DNA sequence.
- ADE HN6A - THY 04 2.12 (+/-0.22) 2.09 (+/-0.19) ADE N7 - THY H3 1.94 (+/-0.16) 1.92 (+/-0.12) Averaged Watson - Crick and Hoogsteen hydrogen bond distances (in Angstroms) in T,AT 2 and T ⁇ T ⁇ helices. These distances are calculated for the triple helical DNA complexes. The fluctuation in atomic position (calculated as the root-mean-square [rms]) are in (A).
- Intrastrand phosphate distances of T ⁇ T j and T ⁇ T-MP helices The calculated intrastrand phosphate distances (in Angstroms) averaged over the 40 psec trajectory are shown for the entire triple helical systems. Standard interstrand phosphorus distances are 6.0A for A-DNA and 7.0A for B-DNA (15) .
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Abstract
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US36802789A | 1989-06-19 | 1989-06-19 | |
US368027 | 1989-06-19 |
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US5824796A (en) * | 1988-09-28 | 1998-10-20 | Epoch Pharmaceuticals, Inc. | Cross-linking oligonucleotides |
USRE38416E1 (en) | 1988-09-28 | 2004-02-03 | Epoch Biosciences, Inc. | Cross-linking oligonucleotides |
US5849482A (en) * | 1988-09-28 | 1998-12-15 | Epoch Pharmaceuticals, Inc. | Crosslinking oligonucleotides |
CA2083719A1 (en) * | 1990-05-25 | 1991-11-26 | Mark D. Matteucci | "sequence-specific non-photoactivated crosslinking agents which bind to the major groove of duplex dna |
WO1992011390A1 (en) * | 1990-12-17 | 1992-07-09 | Idexx Laboratories, Inc. | Nucleic acid sequence detection by triple helix formation |
US6136601A (en) * | 1991-08-21 | 2000-10-24 | Epoch Pharmaceuticals, Inc. | Targeted mutagenesis in living cells using modified oligonucleotides |
CA2116343A1 (en) * | 1991-08-30 | 1993-03-18 | Paul S. Miller | Formation of triple helix complexes of double stranded dna using nucleoside oligomers which comprise purine base analogs |
IL103311A0 (en) * | 1991-10-07 | 1993-03-15 | Univ Johns Hopkins | Formation of triple helix complexes of single stranded nucleic acids using nucleoside oligomers |
EP0637965B1 (en) * | 1991-11-26 | 2002-10-16 | Isis Pharmaceuticals, Inc. | Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines |
US6235887B1 (en) | 1991-11-26 | 2001-05-22 | Isis Pharmaceuticals, Inc. | Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines |
TW393513B (en) * | 1991-11-26 | 2000-06-11 | Isis Pharmaceuticals Inc | Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines |
US5424413A (en) * | 1992-01-22 | 1995-06-13 | Gen-Probe Incorporated | Branched nucleic acid probes |
US5616461A (en) * | 1992-05-14 | 1997-04-01 | Dana-Farber Cancer Institute | Assay for antiviral activity using complex of herpesvirus origin of replication and cellular protein |
AU6296294A (en) * | 1993-01-26 | 1994-08-15 | Microprobe Corporation | Bifunctional crosslinking oligonucleotides adapted for linking to a desired gene sequence of invading organism or cell |
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US4757055A (en) * | 1980-11-12 | 1988-07-12 | The Johns Hopkins University | Method for selectively controlling unwanted expression or function of foreign nucleic acids in animal or mammalian cells |
US4511713A (en) * | 1980-11-12 | 1985-04-16 | The Johns Hopkins University | Process for selectively controlling unwanted expression or function of foreign nucleic acids in animal or mammalian cells |
US4469863A (en) * | 1980-11-12 | 1984-09-04 | Ts O Paul O P | Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof |
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Non-Patent Citations (6)
Title |
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JOURNAL OF THE AMERICAN CHEMICAL SOCIETY vol. 111, May 1989, WASHINGTON, DC US pages 3059 - 3061 T. J. POVSIC ET AL. 'Triple helix formation by oligonucleotides on DNA extended to the physiological pH range' * |
NUCLEIC ACIDS RESEARCH vol. 15, no. 19, 1987, ARLINGTON, VIRGINIA US pages 7749 - 7759 T. L. DOAN ET AL. 'Sequence specific recognition, photocrosslinking and cleavage of the DNA double helix by an oligo-alpha-thymidilate covalently linked to an azidoproflavine derivative' * |
PROC. NATL. ACAD. SCI. U. S. A., 85(11), 3781-5 1988, KOHWI, YOSHINORI ET AL. 'Magnesium ion-dependent triple - helix structure formed by homopurine-homopyrimidine sequences in supercoiled plasmid' * |
PROC. NATL. ACAD. SCI. U. S. A., 85(5), 1349-53 1988, PRASEUTH, DANIELE ET AL. 'Sequence-specific binding and photocrosslinking of .alpha. and .beta. oligodeoxynucleotides to the major groove of DNA via' * |
SCIENCE (WASHINGTON, D. C., 238 (4827), 645-50 1987, MOSER, HEINZ E. ET AL. 'Sequence-specific cleavage of double helical DNA by triple helix formation' * |
Science ,vol 241, 1988, 456-459, Cooney et al. * |
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AU7584594A (en) | 1994-12-15 |
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CA2019156A1 (en) | 1990-12-19 |
AU679111B2 (en) | 1997-06-19 |
NZ234082A (en) | 1997-06-24 |
WO1990015884A1 (en) | 1990-12-27 |
IL94752A (en) | 1995-12-31 |
AU651067B2 (en) | 1994-07-14 |
AU5846890A (en) | 1991-01-08 |
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