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  • C12N2310/35—Nature of the modification
  • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
  • C12N2310/3521—Methyl

Definitions

• the present invention is directed to novel methods of detecting and recognizing specific sequences in single stranded nucleic acids, particularly RNA, using first and second nucleoside Oligomers which are capable of specifically complexing with a selected single stranded nucleic acid structure to give a triple helix structure.
• Triple helical complexes containing cytosine and thymidine on the 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 N-3 of the pyrimidine ring for hydrogen bonding. Accordingly, in some circumstances, cytosine may be protonated at N-3.
• DNA exhibits a wide range of polymorphic conformations, such conformations may be essential for biological processes. Modulation of signal transduction by sequencespecific protein-DNA binding and molecular interactions such as transcription, translation, and replication, are believed to be dependent upon DNA conformation.
• 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 are reported to be able to 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.
• U.S. Patent No. 4,469,863 and 4,511,713. 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 phosphorothioate 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.” (6)
• Psoralen-derivatized oligonucleoside methylphosphonates have been reported capable of cross-linking either coding or non-coding single stranded DNA; however, double stranded DNA was not cross-linked.
• the present invention is directed to methods of detecting or recognizing a specific segment of single stranded nucleic acid or a single stranded nucleic acid sequence and to methods of preventing expression of function of a specific segment of single stranded nucleic acid having a given target sequence by forming a triple helix complex.
• Triple helix complexes of either DNA or RNA target sequences may be formed.
• the present invention is also directed to novel modified Oligomers which are useful for preventing expression and/or functioning of a selected single-stranded nucleic acid sequence and which optionally include a nucleic acid modifying group. Additionally, the present invention is directed to Oligomers which comprise pyrimidine and/or purine nucleoside analogs. In parti-cular, these purine nucleoside analogs are modified to favor formation and stability of the triplex structure and to decrease misreading of the target nucleic acid sequences.
• the present invention is directed to methods of preventing function or expression of a single stranded nucleic acid target sequence which comprises contacting said target sequence with a first Oligomer and a second Oligomer wherein the nucleoside sequences of said first and second Oligomers are selected so that a triple helix structure is formed.
• the nucleic acid target sequence comprises either a polypurine or a polypyrimidine sequence.
• the first and second Oligomers may comprise only pyrimidine nucleosides or analogs or alternatively one of said first and second Oligomers comprises only purine nucleosides and the other comprises only pyrimidine nucleosides.
• the first and second Oligomers may comprise only purine nucleosides or, alternatively, one of the first and second Oligomers comprises only purine nucleosides and the other comprises only pyrimidine nucleosides.
• analogs of the naturally occurring purine nucleosides are employed in the Oligomers of the present invention. These purine nucleoside analogs have been modified to favor hydrogen bonding configurations which encourage triplex formation and also triplex stability while disfavoring misreading (or misparing) of the target sequences by the bases of the first and second Oligomers and also disfavoring nonselective interactions between the bases on the three nucleic acid strands.
• the present invention is directed to methods of detecting or recognizing a specific segment of single stranded nucleic acid or single stranded nucleic acid sequence and to methods of preventing expression or function of a specific segment of single stranded nucleic acid having a given sequence, especially RNA, by forming a triple helix structure.
• the present invention is also directed to novel modified Oligomers which are useful for preventing expression and/or functioning of a selected double nucleic acid sequence and which optionally include a DNA modifying group. Additionally, the present invention is directed to novel Oligomers which comprise cytosine analogs.
• the present invention is also directed to formation of a triple helix structure by the interaction of a specific segment of single stranded nucleic acid and first and second Oligomers.
• the first Oligomer is sufficiently com plementary to the segment of single stranded nucleic acid to hybridize to it and the second Oligomer is sufficiently complementary to the hybrid to read it and base pair (or hybridize) thereto.
• the present invention is directed to methods of detecting or recognizing a specific segment of single stranded nucleic acid which comprises forming a hybrid between the segment and a first oligomer and contacting said hybrid nucleic acid with a second Oligomer which is sufficiently complementary to the sequence of purine bases in said hybrid 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 a single stranded nucleic acid having a given sequence which comprises first forming a hybrid using a complementary first oligomer and then contacting said hybrid with a second Oligomer sufficiently complementary to said double stranded hybrid to form hydrogen bonds therewith, thereby giving a triple helix structure.
• the present invention is directed to methods wherein the single stranded nucleic acid segment comprises an mRNA in a living cell and wherein formation of the triple helix structure inhibits or inactivates said mRNA and prevents its translation.
• the present invention provides methods of preventing or interfering with expression of a single stranded nucleic acid target sequence in vivo where the target sequence is an RNA region which codes for an initiator codon, a polyadenylation region, an mRNA cap site or a splice junction by formation of a triple stranded helix.
• First and second Oligomers are selected that they will form a triple stranded helix and, thus, substantially prevent or interfere with expression of the target sequence.
• the present invention provides methods of selectively preventing or interfering with expression of a gene in a cell or a protein product of a gene by preventing splicing of a pre-mRNA to give a translatable mRNA.
• first and second Oligomers are selected so that they will form a triple stranded helix with the target sequence and introduced into the cell and form a triple stranded helix with the target sequence and prevent splicing of the pre-mRNA.
• the present invention provides methods of inhibiting replication or translation of a single stranded DNA target sequence in vivo without substantially inhibiting overall DNA synthesis by formation of a triple stranded helix by first and second Oligomers with the target.
• said second Oligomer is modified to incorporate a nucleic acid modifying group which, after the second Oligomer hydrogen bonds or hybridizes with the hybrid, is caused to react chemically with the hybrid and irreversibly modify it.
• modifications may include cross-linking second Oligomer and hybrid by forming a covalent bond thereto, alkylating the hybrid, cleaving said hybrid at a specific location, or by degrading or destroying the hybrid.
• the present invention also provides Oligomers which include 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.
• the present invention provides Oligomers wherein a purine analog of adenine or guanine replaces at least one adenine or guanine base.
• 2-amino purine may replace adenine and guanine may be replaced by its 6-selenium analog or by 6-isopropyledine-7-deazaguanine.
• the substitution of at least one 2-aminopurine for a 6-aminopurine (adenine) will provide a favorable regularity in stacking with the guanine base if homopurines are used for the third strand.
• the base patterns of 2-aminopurines and guanines will be isomoiphic and will have the same geometrical pattern.
• the 2-amino group of 2-aminopurine will be serving as a proton donor to N 7 while N 1 of the 2-aminopurine will be serving as a proton acceptor to the 6-amino group of the adenine in the duplex, respectively.
• the 2-amino group of the guanine in the third strand will be a proton donor to the N 7 of the guanine in the duplex and the N 1 proton of the guanine in the third strand will also be a proton donor to the 6-oxo- group of the guanine in the duplex.
• the 2-amino purine (of the third strand) will be donating one proton and accepting one proton, while the guanine in the third strand will be donating both protons to form hydrogen bonds.
• the third strand will have a polarity parallel to the purine strand of the duplex and both nucleosides will be in the anti conformation.
• the replacement of the 6-oxo group in guanine by the 6-selenium group or the isopropylidene group will reduce the non-selectivity of guanine in the third strand for reading other bases.
• first and second Oligomers are provided that comprise at least one nucleosidyl unit having a modified purine base.
• These Oligomers comprise nucleosidyl units (or nucleoside monomers) which may be linked by any one of a variety of internucleosidyl linkages.
• internucleosidyl linkages include, but are not limited to, phosphorus-containing linkages such as phosphodiester linkages, alkyl and aryl-phosphonate linkages, phosphorothioate linkages, phosphoramidite linkages and neutral phosphate ester linkages such as phosphotries ter linkages; as well as internucleosidyl linkages which do not include phosphorus, such as morpholino linkages, formacetal linkages, sulfamate linkages, and carbamate linkages. Other internucleosidyl linkages known in the art may be used in these Oligomers.
• these Oligomers may incorporate nucleosidyl units having modified sugar moieties which include ribosyl moieties, deoxyribosyl moieties and modified ribosyl moieties such as 2'-O-alkylribosyl (alkyl of 1 to 10 carbon atoms), 2'-O-arylribosyl, and 2'-halogen ribosyl, all optionally substituted with halogen, alkyl and aryl, and in particular, 2'-O-methylribosyl moieties.
• modified sugar moieties which include ribosyl moieties, deoxyribosyl moieties and modified ribosyl moieties such as 2'-O-alkylribosyl (alkyl of 1 to 10 carbon atoms), 2'-O-arylribosyl, and 2'-halogen ribosyl, all optionally substituted with halogen, alkyl and
• incorporation of nucleosidyl units having modified ribosyl, particularly 2'-O-methyl ribosyl, moieties may advantageously improve hybridization with the double stranded nucleic acid sequence and also improve resistance to enzymatic degradation.
• the present invention provides novel nonionic alkyl- and aryl-phosphonate Oligomers com- prising these purine nucleoside analogs which are sufficiently complementary to the sequence of a specific single stranded nucleic acid. Segment to hydrogen bond and form a triple helix structure.
• Preferred are nonionic methyl phosphonate Oligomers.
• 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 (such as pseudoisocytosine).
• cytosine analogs include 5-methylcytosine, as well as the analogs depicted in Table V. Definitions
• 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-position, or to the guanine analogs modified at the 6-position or the analog of adenine, 2-amino purine.
• nucleoside includes a nucleosidyl unit and it used interchangeably therewith, and refers to a subunit of a nucleic acid which comprises a 5 carbon sugar and a nitrogen-containing base.
• the term 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 5 carbon sugar is ribose; in DNA, it is a 2'-deoxyribose.
• the term also includes analogs of such subunits, including modified sugars such as 2'-O-alkyl ribose.
• 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” configuration.
• Phosphonate groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
• phosphodiester refers to the group , wherein phosphodiester groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
• a "non-nucleoside monomeric unit” refers to a monomeric unit which does not significantly participate in hybridization of an Oligomer to a target sequence. Such monomeric units must not, for example, participate in any significant hydrogen bonding with a nucleoside, and would exclude monomeric units having as a component, one of the 5 nucleotide bases or analogs thereof.
• nucleoside/non-nucleoside polymer refers to a polymer comprised of nucleoside and non-nucleoside monomeric units.
• oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 6 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means.
• the term "Oligomer” refers to a chain of oligonucleosides which have internucleosidyl linkages linking the nucleoside monomers and, thus, includes oligonucleotides, nonionic oligonucleoside alkyland aryl-phosphonate analogs, alkyl- and aryl-phosphonothioates, phosphorothioate or phosphorodithioate analogs of oligonucleotides, phosphoramidate analogs of oligonucleotides, neutral phosphate ester oligonucleoside analogs, such as phosphotriesters and other oligonucleoside analogs and modified oligonucleosides, and also includes nucleoside/non-nucleoside polymers.
• nucleoside/non-nucleoside polymers wherein one or more of the phosphorus group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a morpholino linkage, a formacetal linkage, a sulfamate linkage or a carbamate linkage.
• a non-phosphorous linkage such as a morpholino linkage, a formacetal linkage, a sulfamate linkage or a carbamate linkage.
• alkyl- or aryl-phosphonate Oligomer refers to Oligomers having at least one alkyl- or aryl-phosphonate internucleosidyl linkage.
• methylphosphonate Oligomer (or “MP-Oligomer”) refers to Oligomers having at least one methylphosphonate internucleosidyl linkage.
• neutral Oligomer refers to Oligomers which have nonionic internucleosidyl linkages between nucleoside monomers (i.e., linkages having no net positive or negative ionic charge) and include, for example, Oligomers having internucleosidyl linkages such as alkyl- or arylphosphonate linkages, alkyl- or aryl-phosphonothioates, neutral phosphate ester linkages such as phosphotriester linkages, especially neutral ethyltriester linkages; and non-phosphorus-containing internucleosidyl linkages, such as sulfamate, morpholino, formacetal and carbamate linkages.
• internucleosidyl linkages such as alkyl- or arylphosphonate linkages, alkyl- or aryl-phosphonothioates
• neutral phosphate ester linkages such as phosphotriester linkages, especially neutral ethyltriester linkages
• a neutral Oligomer may comprise a conjugate between an oligonucleoside or nucleoside/non- nucleoside polymer and a second molecule which comprises a conjugation partner.
• conjugation partners may comprise intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, photo-cross-linking agents such as psoralen, and the like.
• conjugation partners may further enhance the uptake of the Oligomer, modify the interaction of the Oligomer with the target sequence, or alter the pharmacokinetic distribution of the Oligonucleoside.
• the essential requirement is that the oligonucleoside or nucleoside/non-nucleoside polymer that the conjugate comprises be neutral.
• neutral alkyl- or aryl-phosphonate Oligomer refers to neutral oligomers having neutral internucleosidyl linkages which comprise at least one alkyl- or aryl-phosphonate linkage.
• neutral methylphosphonate Oligomer refers to neutral Oligomers having internucleosidyl linkages which comprise at least one methylphosphonate linkage.
• Triplex Oligomer Pair refers to first and second Oligomers which are capable of reading a segment of a single stranded nucleic acid, such as RNA or DNA, and forming a triple helix structure therewith.
• Third Strand Oligomer refers to Oligomers which are capable of reading a segment of a double stranded nucleic acid, such as a DNA duplex or a DNA-RNA duplex, and forming a triple helix structure therewith.
• Complementary when referring to a Triple Oligomer Pair or first and second Oligomers (or to a Third Strand Oligomer), refers to Oligomers having base sequences which hydrogen bond (and base pair or hybridize) with the base sequence of a single stranded nucleic acid (or to a nucleic acid duplex for the Third Strand Oligomer) to form a triple helix structure.
• p in, e.g., as in ApA represents a phosphodiester linkage
• p in, e.g., as in CpG represents a methylphosphonate linkage
• T indicates nucleosidyl groups linked by methyl phosphonate linkages.
• read refers to the ability of a nucleic acid residue to recognize through hydrogen bond interactions and base sequence of another nucleic acid.
• a corresponding base of each Triple Oligomer pair Oligomer is able to recognize through hydrogen bond interactions the corresponding base of the segment of a single stranded DNA and form a triplet therewith.
• triplet refers to a situation such as that depicted in Figures 1A, 1B; 2A to 2D and 3A to 3C, wherein a base of each of the first and second Oligomers has hydrogen bonded (and thus base paired) with the corresponding base of the target segment of single stranded DNA or RNA.
• Figures 1A and IB depict triplets wherein two pyrimidine bases forms a triplet with a central purine base.
• Figures 2A to 2D depict triplets wherein a purine base and a pyrimidine base forms a triplet with a central purine base.
• Figures 3A to 3C depict triplets incorporating analogs of G or A.
• Figures 4A to 4E depict the base sequences of exemplary mixed sequence triple helix structures wherein one of the Triplex Oligomer pair "reads" across the two other strands and, thus, base pairs with purine bases on both other strands.
• Figure 5 depicts a nucleosidyl unit having a modified sugar moiety with an alkyleneoxy link for lengthening internucleoside phosphorus linkages and processes for its preparation.
• Figure 6 depicts a nucleosidyl unit having a modified sugar moiety with an alkylene link for lengthening internucleoside phosphorus linkages and processes for its synthesis.
• Figure 7 depicts CD spectra of triple helix structures, (—) depicts a MP-Oligomer Third Strand, ( ⁇ ) depicts an Oligonucleotide Third Strand.
• Figures 8A and 8B depict cross-linking of (A) single stranded DNA and (B) double stranded DNA using psoralenderivatized MP-Oligomers.
• Figures 9A to 9E depict CD spectra.
• Figures 9A depicts CD spectra of oligomers I (-) and II ( ⁇ ) in buffer A in room temperature and III (-•-) in buffer B at room temperature.
• Figure 9B depicts CD spectra of I-II, 2:1 at room temperature in buffer A (-) and buffer B
• FIG. 9E depicts CD spectra of II-III (1:1) + I in buffer A at room temperature (-) and at 3 °C (- - - - - ) and in buffer C at room temperature ( ⁇ ) and at 3°C ( ⁇ - ⁇ ).
• Figure 10 depicts autoradiograms of polyacrylamide gel electrophoresis patterns: Lane 1, II; Lane 2, III-II (2:1); Lane 3, I-II (2:1); Lane 4, III; Lane 5, III-II (1.5:1); Lane 6 I-II-III (1.5:1:1.5).
• Figures 11A and 11B depict UV melting/annealing profiles of I-II-I.
• Figure 11A depicts the normalized hyperchromity changes and
• Figure 11B the ratio of the hyperchromicity changes, for dissociation (-) and association ( ⁇ ).
• Figure 12 depicts CD spectra of 2'-O-methyl (piCU) 8 . r(AG) 8 , 2:1 (—), and 2'-O-methyl (piCU) 8 d(AG) 8 ( ⁇ ) at room temperature.
• Figure 13 depicts a melting curve for 2'-O-methyl (piCU) 8 -r(AG) 8 (2:1).
• Figures 14A and 14B depict breakpoints in mixing curves for d(AG) 8 + d(CT) 8 and d(AG) 8 + d(CT) 8 .
• Figures 15A-15D depict CD spectra.
• Figure 15A depicts CD spectra for d(AG) 8 -d(CT) 8 , 1:1 (-) and d(AG) 8 single strand ( ⁇ ).
• Figure 15B depicts d(AG) 8 -d(CT) 8 , 2:1
• FIG. 15C depicts CD spectra for d(AG) 8 -d(CT) 8 , 1:1 (-), d(AG) 8 single strand ( ⁇ ), d(CT)8 single strand (••••).
• Figure 15D depicts CD spectra for d(AG) 8 ⁇ d(CT) 8, 2:1 (-) and calculated spectrum from a weighted average of duplex and single strand ( ⁇ ).
• Figures 16A-16C depict thermal denaturation curves for d(AG) 8 ⁇ d(CT) 8 , 1:1, (16A); d(AG) 8 ⁇ d(CT) 8 , 1:1 (16B) and d(AG) 8 ⁇ d(CT) 8 , 2:1, (16C).
• Figure 17 depicts an autoradiograph of a native polyacrylamide gel, having gamma [ 32 P] end labelled d(CT) 8 , (lanes 1 to 12) and d(AG) 8 (lanes 13 to 17).
• Figure 18A depicts hydrogen bonded NH-N resonances for d(AG) 8 ⁇ d(CT) 8 mixtures, 1:1 and 2:1.
• Figure 18B depicts temperature dependence of the chemical shift for the three resonances observed for the 2:1 mixture of Figure 18A.
• the present invention involves the formation of triple helix structures with a selected target single stranded nucleic acid sequence by contacting said nucleic acid with a first and second Oligomers, the Triplex Oligomer Pair, which are selected having nucleoside sequences such that they form a triple helical (or helix) complex with the target sequence.
• this invention includes the following aspects.
• a first aspect concerns the reading (or recognition) of the bases in a single stranded nucleic acid segment, through hydrogen bond formation by the bases in the first and second Oligomers using the extra hydrogen bond sites of purines such as adenine and guanine or analogs thereof.
• purines such as adenine and guanine or analogs thereof.
• central purine in reading the base sequence in the single stranded nucleic acid to give a triplet, there is always a purine in the central position of the triplet ("central purine").
• the central purine base may be on the single stranded target sequence, or if the target has a pyrimidine base, it will be on one of the first or second Oligomers.
• the triplet is formed through hydrogen bond formation with the bases in the other two strands with the remaining available hydrogen bonding sites of the central purine base.
• Either purines such as adenine (a), guanine (b) or the adenine and guanine analogs described herein), or pyrimidines (thymine (T), cytosine (c) or cytosine analogs described herein may form hydrogen bonds with the central purine.
• Adenine (A) in the central position of the triplet is read or hydrogen bonded with A (or an A analog) or T in the other strands.
• Guanine (G) in the central position of the triplet is read or hydrogen bonded with C (or a C analog) or G (or a G analog) in the other strands.
• the phosphorus-containing backbone of the first and second Oligomers comprise methylphosphonate groups as well as naturally occurring phosphodiester groups.
• the base planes of the purines and pyrimidines are rigid, and the furanose ring only allows a small ripple
• the conformational state of the nucleoside is defined principally 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'-1 to N-9 or N-1 bond.
• the sugar-base torsion angle, ⁇ CN has been defined as "the angle formed by the trace of the plane of the base with the projection of the C-1' to 0-1' bond of the furanose ring when viewed along the C'-1 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-1' to N.”
• This angle has also been termed the glycosyl torsion angle.
• ⁇ CN for the nucleosides
• 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 nucleosidyl 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 conformation 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.
• DNA duplex having the same polarity of the Watson strand from 5' to 3' would be as follows: (the Third Strand parallel to the Watson 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 nucleic acid.
• 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 second purine is located in the opposite anti-Parallel ("Crick") strand (now the opposite strand is anti-parallel to the Third Strand)
• the purine nucleosidyl 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 nucleic acid.
• the distance between nucleosidyl units along the phosphorus backbone must be increased.
• Two 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 lengthening 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 "nucleosidyl unit one" to the 3' oxygen of the subsequent "nucleosidyl unit two” may be increased by two atoms (such as -CH 2 CH 2 -) or by 3 atoms (such as -O-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'- carbon of its neighbor, would be employed to read the purine bases located on opposite strands.
• Such a non-uniform lengthening link format would be particularly suitable for use in Third Strands comprising both pyrimidine (or pyrimidine analog) and purine bases.
• Figure 1A depicts a triplet having a central A base which is hydrogen bonded to a T on either side.
• one T-containing strand is aligned parallel to the A-containing strand and the other T-containing strand is aligned antiparallel to the A-containing strand. Accordingly, the sequence for that triplet is written as follows.
• Figure 1B depicts a triplet having a central G which is hydrogen bonded to a protonated C on one side and a C on the other side.
• one of the Triplex Oligomer pair has a cytosine protonated at N3 in order to form hydrogen bonds necessary for a stable triplet.
• that base may be replaced by a cytosine analog having a nitrogen bearing a proton at a position analogous to N-3.
• the strand containing C+ (or its analog) is aligned parallel to the central G-containing strand.
• the C-containing strand is aligned antiparallel to the central G-containing strand.
• Figure 2A depicts a triplet wherein the central A forms a hydrogen bond with an A on one side ("side A") and a T on the other.
• the strand containing the side A is aligned parallel to the strand containing the central A.
• the strand containing the T is aligned anti-parallel to the strand containing the central A.
• the glycosyl (C-N) torsion angle of the side A is in the syn conformation and the glycosyl torsion angles of the central A and the T bases are both in the anti conformation.
• Figure 2B also depicts a triplet where a central A forms a triplet with an A one side and a T on the other.
• the strand containing the side A is aligned anti-parallel to the strand containing the central A.
• the strand containing the T is aligned parallel to the strand containing the central A.
• all three bases are in the anti conformation,
• Such a triplet sequence is written as follows:
• Figure 2C depicts a triplet having a central G hydrogen bonded to a G on one side ("side G") and a C on the other.
• the strand containing the side G is aligned parallel to the strand containing the central G and the strand containing the C is aligned antiparallel to the strand containing the central G.
• the glycosyl torsion angle of the side G is in the syn conformation and the glycosyl torsion angles of the central G and the C are both in the anti conformation.
• Figure 2D also depicts a triplet where a central G is hydrogen bonded to a G on one side and a C on the other side.
• the strand containing the side G is aligned anti-parallel to the strand containing the central G and the strand containing the C is aligned anti-parallel to the strand containing the central G.
• the glycosyl torsion angles for all three bases are in the anti conformation,
• Such a triplet sequence is written as follows:
• Figures 3A and 30 depict a triplet wherein a central G is hydrogen bonded to a modified G on one side and a C on the other side.
• the strand containing the modified G either 2-amino-9- ⁇ -D-ribofuranosyl purin-6-selene ("6-selenium guanosine) in Figure 3A or 6-isopropyledene- 7-deazaguanosine in Figure 3B is aligned anti-parallel to the strand, containing the central G and the strand containing the C is aligned anti-parallel to the strand containing the central G.
• the triplet containing the 6-selenium guanosine is written as follows:
• Figure 3C depicts a triplet wherein a central A IS hydrogen bonded to a modified A (2-amino purine) on one side and a T on the other side.
• the strand containing the 2-amino-purine is aligned parallel to the strand containing the central A and the strand containing the T is aligned anti parallel to the strand containing the central A.
• Such a triplet sequence is written as follows:
• N 1 of 2-aminopurine is accepting a proton from the 6-amino group of the central A and that the 2-amino group of the 2-aminopurine is donating a proton to the N 7 of the central A.
• This arrangement will induce the guanine base in the third strand to form a hydrogen bond pair by donating a proton from both N, H and the 2-amino group to the 6-oxo group and N 7 group of the central G in a successive triplet.
• a to A pairing should contain a donor and a receptor arrangement from the third strand of A and the G to G pairing should contain both donor arrangements from the third strand of G.
• the single stranded nucleic acid comprises a polypurine sequence
• it can form triplets where that strand provides the central purine of the triplets formed, so that the target is completely sandwiched by the first and second Oligomers.
• This Triplex Oligomer pair may both comprise having complementary polypyrimidine sequences or alternatively one of the first and second Oligomers has a polypurine sequence and one has a polypyrimidine sequence.
• the single stranded target nucleic acid comprises a polypyrimidine sequence
• that target nucleic acid will not provide the central purine bases for triplet formation.
• the first and second Oligomers which form the triple helix structure with the single stranded target may both comprise polypyrimidine sequence or alternatively, one of the first and second Oligomers may have a polypurine sequence and the other a polypurine sequence. Since the pyrimidine bases of the target may not provide the central base of the triplets, an "open sandwich" triple helix complex is formed wherein one of the Triplex Oligomer Pairs provides the central purine base of the triplet. In order to decrease formation of duplex between the Triplet Oligomer Pairs, it may be preferred that the first and second Oligomers both comprise polypurine sequences.
• Mixed sequences are sequences wherein the selected single stranded nucleic acid target sequence comprises both pyrimidine and purine bases.
• the triplets formed using the first and second Oligomers requires a central purine base that can hydrogen bond to two other bases to form a triplet. Accordingly, one of the first and second Oligomers must be able to "read” across the other two strands. Examples of mixed sequences including appropriately first and second Oligomers are depicted in Figures 4A to 4E.
• Figure 4A depicts a mixed sequence wherein the Oligomer which reads across the other two strands is pyrimidine-rich.
• Figure 4B depicts a mixed sequence wherein the Oligomer which reads across the other two strands is purine-rich.
• Figures 4C, 4D and 4E depict mixed sequences which reads across the Oligomer containing only purine bases.
• "s” denotes syn and "a” denotes anti; no superscript denotes anti.
• first and second Oligomers must read two strands across from one strand to the opposite strand in order to base pair with a central purine base
• 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 other strands to read consecutive central purine bases on the same strand.
• Oligomers which are capable of reading central purine bases either of the single stranded target or of the other Oligomer, may be prepared.
• first and second Oligomers having at least about 7 nucleosides, which can be a sufficient number to allow for specific binding to a desired sequence of a selected target segment of single stranded DNA or RNA. More preferred are Oligomers having from about 8 to about 40 nucleosides; especially preferred are Oligomers having from about 10 to about 25 nucleosides. Due to a combina tion of ease in synthesis, specificity for a selected target sequence, coupled with minimization of intraOligomer, and internucleoside interactions such as folding and coiling, it is believed that Oligomers having from about 12 to about 20 nucleosides comprise a particularly preferred group.
• Oligomers may comprise either ribosyl moieties or deoxyribosyl moieties or modifications thereof. However, due to their easier synthesis and increased stability, Oligomers comprising deoxyribosyl or modified ribosyl moieties (such as 2'-O- methyl ribosyl 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, phosphoro-amidate analogs and neutral phosphate triester oligonucleoside analogs.
• oligonucleoside alkyl- and aryl-phosphonate analogs in which phosphonate linkages replace one or more of the phosphodiester linkages which connect two nucleosidyl units.
• MP-Oligomers Preferred synthetic methods for methylphosphate Oligomers are described in Lee, B.L., et al., Biochemistry 17:3197-3203 (1988), and Miller, P.S., et al., Biochemistry 25: 5092-5097 (1986), the disclosures of which are incorporated herein by reference.
• oligonucleosidyl alkyl- and arylphosphonate analogs wherein at least one of the phosphodiester internucleoside linkages is replaced by a 3'-5' linked internucleoside methylphosphonyl (MP) group (or "methylphosphonate”).
• MP internucleoside methylphosphonyl
• the methylphosphonate linkage has a bond length similar to the bond length of the phosphate groups of oligonucleotides.
• MP-oligomers should present minimal steric restrictions in the interaction with selected nucleic acid sequences.
• MP-Oligomers should be very 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 recognition of the desired segment of single stranded DNA or RNA. 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. Due to a combination of ease of preparation, with specificity for a selected sequence and minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, particularly preferred are MP-Oligomers of from about 12 to 20 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-Oligomer permits kinase labelling and electrophoresis of the Oligomer and also improves its solubility.
• the selected single stranded nucleic acid 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 single stranded nucleic acid sequence in a mixture of nucleic acids or in samples including isolated cells, tissue samples or bodily fluids.
• These 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.
• labelling groups such as psoralen, chemiluminescent groups, cross-linking agents, inter-calating 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 EDTA-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 14 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.
• hybridization 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, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands.
• One preferred labelling method includes incorporation of acridinium esters.
• Such labelled Oligomers are particularly suited as hybridization assay probes and for use in hybridization assays.
• one or both of these Oligomers may be advantageously derivatized or modified to incorporate a nucleic acid modifying group which may be caused to react with said nucleic acid and irreversibly modify its structure, thereby rendering it non-functional.
• a nucleic acid modifying group which may be caused to react with said nucleic acid and irreversibly modify its structure, thereby rendering 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.
• nucleic acid modifying groups may be used to derivatize these Oligomers.
• Nucleic acid modifying groups include groups which, after the derivatized Oligomer (or Oligomers) forms a triple helix structure with the single stranded nucleic acid segment, may be caused to form a covalent linkage, cross-link, alkylate, cleave, degrade, or otherwise inactivate or destroy the nucleic acid segment or a target sequence portion thereof, and thereby irreversibly inhibit the function and/or expression of that nucleic acid segment.
• nucleic acid modifying groups in the Oligomer may be varied and may depend on the particular nucleic acid modifying group employed and the targeted single stranded nucleic acid segment. Accordingly, the nucleic acid modifying group may be positioned at the end of the Oligomer or intermediate the ends. A plurality of nucleic acid modifying groups may be included. Also both of the first and second Oligomers may include nucleic acid modifying groups.
• the nucleic acid modifying group is photoreactable (e.g., activated by a particular wavelength, or range of wavelengths of light), so as to cause reaction and, thus, cross-linking between the Oligomer and the single stranded nucleic acid.
• nucleic acid modifying groups which may cause cross-linking are the psoralens, such as an aminomethyltrimethyl psoralen group (AMT).
• AMT aminomethyltrimethyl psoralen group
• the AMT is advantageously photoreactable, and thus must be activated by exposure to particular wavelength light before cross- linking is effectuated.
• Other cross-linking groups which may or may not be photoreactable may be used to derivatize these Oligomers.
• the nucleic acid modifying groups may comprise an alkylating agent group which, on reaction, separates from the Oligomer and is covalently bonded to the nucleic acid segment to render it inactive.
• alkylating agent groups are known in the chemical arts and include groups derived from alkyl halides, haloacetamides, and the like.
• Nucleic acid modifying groups which may be caused to cleave the nucleic acid segment include 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 bleomycin, 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 a first or second Oligomer which read purine bases on two strands to form a triplex sequence may comprise a mixture of purine and pyrimidine bases or only purine bases.
• purine bases on two strands of a triplex sequence are to be read, it is preferred to use a "read across" Oligomer having only purine bases. It is believed that such purine-only Oligomers are advantageous for several reasons: (a) purines have higher stacking properties than pyrimidines, which would tend to increase stability of the resulting 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;
• the purine bases (and pyrimidine bases as well) are normally in the anti conformation; however, the barrier for a base to roll over to the syn conformation is low.
• Third Strand may assume the syn conformation during the hydrogen bonding process. If desired, it is possible the modify the purine so that it is normally in the syn conformation formation.
• the purine may be modified at the
• invention contemplates the optional incorporation of 8-substituted purines in place of unsubstituted A or G. Studies with Kendrew models indicate that such substitutions should not affect formation of the triple helix structure.
• a non-uniform link format is used, as described herein, to allow the third strand ("read across Oligomer") to read across from one of the two strands 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 such that the phosphonate linkage is not sterically hindered and the groups do not interact with each other
• R' is hydrogen, hydroxy or methoxy
• alk is alkylene of 2 to 6 carbpn atoms or alkylene of 2 to 6 carbon atoms.
• Oligomers which comprise at least about 7 nucleosidyl units.
• nucleosidyl units where R is methyl are 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.
• preferred Oligomers which read central purine bases may include purine analogs, modified to favor triplet formation and stability. These purine analogs include 6-selenium guanosine or 6-isopropylidene-7-deazaguanosine in place of guanosine or 2-amino purine in place of adenosine.
• 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 in the duplex 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 in the duplex at neutral pH and include 2'-deoxy-5,6-dihydro-5-azadeoxycytidine (I), pseudoisocytidine (II), 6-amino-3-( ⁇ -D-ribofuranosyl)pyrimidine-2,4-dione (III) and 1-amino-1,2,4-( ⁇ -D-deoxyribofuranosyl)triazine-3-[4H]-one (IV), the structures of which are set forth in Table 5.
• D Preparation of MP-Oligomers
• Oligomers comprising nucleosidyl units which comprise modified sugar moieties having lengthening links (see Figures 5 and 6) may be conveniently 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).
• 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
• 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.
• 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
• Figure 6 depicts a proposed reaction scheme for a
• DCC denotes dicyclohexylcarbodiimide
• DMSO denotes dimethylsulfoxide
• B is a base.
• Suitable protecting groups, R include t-butyldimethyl silyl and tetrahydropyranyl.
• MP-Oligomers incorporating the above-described modified nucleosidyl units are prepared as described above, substituting the modified nucleosidyl unit.
• nucleosidyl units having the same lengthening links may be employed.
• a mixture of nucleosidyl units having no lengthening link and lengthening 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.
• a specific segment of single stranded nucleic acid may be detected or recognized using first and second Oligomers which form a triple helix with the single stranded nucleic acid according to the triplet base pairing guidelines described herein.
• the first and second Oligomers have sequences selected such that a base of each nucleosidyl unit of each Oligomer will form a triplet with a corresponding base of the single stranded nucleic acid target to give a triple helix structure.
• Detectably labeled Oligomers may be used as probes for use in hybridization assays, for example, to detect the presence of a particular single-stranded nucleic acid sequence.
• the present invention also provides a method of preventing expression or function of a selected target sequence of single stranded nucleic acid by use of first and second Oligomers which hydrogen bond with and form a triple stranded helix structure with the single stranded target as described above. Formation of the triple stranded helix may prevent expression and/or function by modes such as preventing transcription, preventing of binding of effector molecules (such as proteins), etc.
• the target seguence of single stranded nucleic acid will be recognized twice, or in two steps, (1) one time by duplex formation by Watson-Crick base pairing with the first Oligomer and (2) a second time by triplex formation with the second Oligomer with or without an internucleosidyl lengthening link between nucleosides of the second Oligomer to allow the second Oligomer to read across strands.
• Derivatized Oligomers may be used to detect or locate and then irreversibly modify the target site in the nucleic acid by cross-linking (psoralens) or cleaving one or both strands (EDTA). By careful selection of a target site for cleavage, one of the Oligomers may be used as a molecular scissors to specifically excise a selected nucleic acid sequence.
• the Oligomers may be derivatized to incorporate a nucleic acid reacting or modifying group which can be caused to react with the nucleic acid segment or a target sequence thereof to irreversibly modify, degrade or destroy the nucleic acid and thus irreversibly inhibit its functions.
• Oligomers may be used to inactivate or inhibit a particular gene or target sequence of the same in a living cell, allowing selective inactivation or inhibition.
• the target sequence may be DNA or RNA, such as a pre-mRNA, an mRNA or an RNA sequence such as an initiator codon, a polyadenylation region, an mRNA cap site or a splice function.
• 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.
• kits for detecting a particular single stranded nucleic acid sequence which comprises first and second Oligomers, at least one of which is a detectably labeled purine MP-Oligomer Third selected to be able sufficiently complementary to the target sequence of the single stranded nucleic acid to be able form a triple helix structure therewith.
• Triple-stranded poly (dT 10 ) -poly (dA 10 ) -poly (dT 10 ) [T 1 AT 2 ] 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 ab initio quantum mechanical methods with QUEST (ver
• 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.
• TIP3P water 14
• the box dimensions were 101, 686.8 A 3 for T 1 AT 2 , and 124,321.1 A 3 for T 1 AT 2 MP.
• 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 1 AT 2 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 2 MP.
• the reduced interstrand phosphorus atom distance and smaller mean atomic fluctua-tions 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 T1 and A strands) averaged 39.4 degrees ( ⁇ -2.86) for the T 1 AT 2 structure and is more consistent with a B-DNA conformation (range 36-45).
• the T 1 AT 2 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 TI and A strands) for the entire structure are for 10.2 T 1 AT 2 and 11.2 degrees for T 1 AT 2 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 2 MP strand are more consistent with a B-DNA conformation, in contrast to values more consistent with A-DNA for the T 2 strand.
• 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 concen-tration (10,17,18).
• the large perturbation of the ⁇ dihedral and variable conformational fluctuation of the R P- and S P- MP diastereoisomers in the triplet are due to nonbonded and hydrophobic interactions.
• the Sp-MP groups are in close proximity to thymine methyl groups (in the major groove) on the same DNA strand, and interact by Van der Waals forces, which could locally destabilize the helix by "locking" the thymine to the Sp-MP backbone.
• Circular dichroism spectroscopy studies were performed using Triple Helix Structures formed using a combination following nucleoside oligomers.
• Figure 7 shows the CD spectra for triple helix (a) [2:1 d(CT) 8 ⁇ d(AG) 8 (— ] and (b) [1:1:1 d(CT) 8 ⁇ d(AG) 8 ⁇ d(CT) 8 ( ⁇ )].
• Psoralen derivatized dTp(T) 6 oligomers were prepared as described in Lee, B.L., et al., Biochemistry 27:3197-3203 (1988).
• the T 7 oligomers were allowed to hybridize with DNA having the following sequence including a 15-mer poly dA 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.
• APE HN6B - THY 04 2.33 (+/-0.31) 1.98 (+/-0.15) ADE N1 - THY H3 2.10 (+/-0.17) 1.95 (+/-0.13) HOOGSTEEN
• Q Averages of sugar pucker (Q), phase, and conformational populations of furanose for T 1 AT 2 and T 1 AT 2 MP helices (15).
• Q is in Angstroms
• phase is in degrees
• populations are in percent of the total furanose conformations in each of the three DNA strands.
• Intrastrand phosphate distances of T 1 AT 2 and T 1 AT 2 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).
• Formation of a triple helix complex using a d(AG) 8 single stranded target (II) was demonstrated using two strands of an oligonucleotide analog containing 2'-O- methyl-pseudoisocytidine (piC) and 2'-O-methyluridine (X) in an alternated sequence (piC X) 7 piCT (I).
• piC 2'-O- methyl-pseudoisocytidine
• X 2'-O-methyluridine
• II alternated sequence
• Formation of a triple helix complex between a d(AG) 8 ⁇ d(CT) 8 duplex (II: III) and I was also demonstrated.
• d(CT) 8 is represented by III.
• Radioactivity was counted on an LS 7500 liquid scintillation counter (Beckman, Columbia, MD).
• buffer A 0.01 M Na phosphate, 0.1 M NaCl, 0.01 mM EDTA p H7.2
• buffer B 0.02 M Na phosphate, 0.01 M NaCl, 5 mM MgCl 2
• buffer C 0.01 M Na phosphate, 0.35 M NaCl, 5 mM MgCl 2 , pH 7.2
• buffer D 0.06 M Tris borate, 5 mM MgCl 2 , 0.075 mM EDTA, pH 7.3.
• X and piC were synthesized according to the reported methods (30, 31, 32), and were converted to their corresponding amidite synthons (33, 34).
• the oligo-nucleotide I was synthesized on a DNA synthesizer (either Milligen 7500 or Applied Biosystem) according to the reported methods (31, 32). After being deblocked and cleaved from the solid support by cone. NH 4 OH treatment, I with protecting dimethoxytrityl (DMTr) groups was purified by HPLC. Fractions were treated with 80% acetic acid solution to deblock the DMTr group after which oligomers were purified by HPLC. The purified oligomer I of this preparation showed a single peak by HPLC analysis. Starting from 2 ⁇ mole solid supporting material 2.5 O.D. units of I were obtained.
• CD spectra were obtained on a J-500A CD spectropolarimeter (Jasco, Japan). Sample temperature was controlled by using a circulating water bath. The Oligomer or the Oligomers were concentrated in vacuo, after which the residues were dissolved in appropriate buffers, except for the preparation of the I-II-III sample. This mixture was prepared by cooling a solution of II-III duplex in buffer A on an ice bath, and then adding this solution to I. The whole solution mixture was kept on an ice bath for 30 minutes, and then kept at room temperature for another 30 minutes. After CD spectra of this mixture were measured, a concentrated salt solution was added to the mixture to set the final salt condition as for buffer C, then other spectra were measured. Sample temperature was controlled by fluid circulating from a temperature regulated water bath.
• UV absorbance was measured by Varian DMS 100 and 219 spectrophotometers. Thermal profiles of melting/annealing was monitored by the Varian 219 with a thermoregulated sample compartment. Sample temperature was controlled by fluid circulating from a temperature regulated bath monitored with a calibrated thermistor probe inserted in a "dummy" cuvette.
• Figure 9A depicts the CD spectra of the single stranded I, II and III at pH 7.2.
• the CD spectrum of I: II (2:1) showed a large negative band at 213 nm in 0.1 M NaCl (buffer A); a similar spectrum was observed upon addition of 0.25 M NaCl and 5 nM MgCl 2 (buffer C).
• Such a spectrum has been shown to be indicative of homopyrimidine-homopurine-homopyrimidine triplex formation for the repeating dinucleotide sequences AG/CT in both polymer and oligomer systems ( Figure 9B) (35, 35).
• Figure 9C depicts the CD spectrum of I:II mixture at one to one ratio.
• Figure 9C does not show such a large negative band in this wavelength range; however, this spectrum is identical to a calculated spectrum derived from the spectrum of one half of single stranded II and the spectrum of one half of I:II (2:1).
• the observed CD spectrum indicates the 1:1 I:II mixture is one half I:II:I triplex and one half single stranded II.
• this CD study indicates that the I:II:I is favored over the I:II duplex, even at stoichiometric ratios which would favor duplex formation.
• the mobility of single-stranded II is shown in lane 1.
• the mixture of II and III shows only one band in spite of a one to two concentration ratio (lane 2).
• the mobility of this band is less than that of II itself.
• Two bands are detected in the mixture of II and I (lane 3) again with a concentration ratio one to two.
• the band with faster mobility can readily be recognized as the II-I duplex.
• the slower band is the evidence of formation of the I-II-I triplex. It is interesting to note that only one band is detected in lane 2, which is clear evidence that the III-II-III triplex is not formed at these conditions.
• Lanes four to six in Figure 10 are the electrophoretic results when single-stranded III is labeled by 32 P at the 5'-end.
• the Mobility of III itself is shown in lane 4 as a reference.
• the mixture of III and II (1.5:1) is shown in lane 5.
• the slower moving band has a comparable mobility to the band observed in lane 2 leaving no doubt as to the formation of the III-II duplex.
• the excess amount of single stranded III is present as a faster moving band which is identical to that in lane 4. This confirms the results obtained from lane 2, namely, that no III-II-III triplex is formed at neutral pH even in the presence of MgCl 2 .
• the triplex I-II-III is observed when a 1.5:1:1.5 mixture was electrophoresed in lane 6.
• the three bands clearly correspond to triplex, duplex, and single strand from top to bottom, respectively, by comparison to bands observed in lanes 2 to 5.
• the bands in lanes 5 and 6 were cut from the gel plate and counted.
• the ratio of radioactivities of duplex as single stranded III are about two to one
• the observed CD spectrum differs from the additive CD spectrum (see Figure 12), especially in the 210 nm region which indicated triple helix formation.
• Figure 14 depicts continuous variation in compositions of UV absorption measured at d (AG) 8 phosphodiester and d(AG) 8 methylphosphonate homopurine oligomers with d(CT) 8 at four wavelengths (•) 280 nm, ( ⁇ ) 260 nm, (D) 254 nm, and (O) 235 nM.
• Total strand concentration was 2.4 ⁇ m in 0.1 M Na + 0.01 M PO 4 -3 , 10 -5 M EDTA, pH 7.0.
• a single end point at 1:1 purine:pyrimidine stoichiometric ratio was observed for the interactions of the phosphodiesters d(AG) 8 and d(CT) 8 which indicated that only a Watson-Crick type duplex formed under these conditions.
• Figure 15 depicts observed CD spectra.
• the observed spectrum for 2:1 d(AG) 8 :d(CT) 8 was very different from the spectrum calculated by simple addition which indicated triple stranded helical complex formation.
• the CD spectra were run at 20°C in 0.1 M Na + , 0.01 M PO 4 -3 , 10 -5 M EDTA, pH 8.0. Total strand concentration was 4.8 ⁇ M and the e was reported per mole of base residue.
• Figure 16 depicts the TT m and melting profiles of d(AG) 8 :d(CT) 8 1:1, d(AG) 8 :d(CT) 8 2:1.
• the thermal denaturation profiles for the Oligomer complexes were monitored by UV absorption hyperchromicity.
• Total strand concentration was 4.8 ⁇ m in 0.1 M Na + , 0.01 M PO 4 -3 , 10 -5 M EDTA, pH 8.0.
• each curve was normalized to the total change in absorbance.
• the Tm for d(AG) 8 :d(CT) 8 1:1 was 50°C.
• the Tm for d(AG) 8 :d(CT) 8 1:1 was 53°C.
• the Tm for d(AG) 8 :d(CT) 8 2:1 was 51°C. As may be seen from the melting curves, the melting profile observed for the triplex was much more narrow or sharper. This date suggested a simultaneous dissociation of duplex and triplex, but that the transition of the triplex was more homogeneous in thermal stability.
• Figure 17 depicts the electrophoretic analysis of the complexes in native polyacrylamide gel.
• Figure 16 is an auto-radiograph of a native 16% (29:1 bis) polyacrylamide gel containing gamma [ 32 P] end labelled d(CT) 8 (lanes 1-12) and d(AG) 8 (lanes 13-17) and their complexes.
• the gel was electrophoresed at four volts per CM for 30 hours at 5°C in 0.1 M NaCl, 0.04 M Tris, 0.01 M PO 4 -3 , 10 -3 M EDTA, pH 8.0 with buffer recirculation to prevent pH changes.
• the concentration of Oligomers in each lane is shown below the stoichiometric ratio of the interacting strands.
• Figure 18A depicts the hydrogen-bonded NH-N resonance of a 1:1 and a 2:1 mixture of d (AG) 8 and d(CT) 8 at 300 MHz in 0.1 M Na+, 0.01 M PO 4 -3 , 10 -5 M EDTA, pH 8.0.
• Figure 17A depicts spectra at 30°C for 1:1 and 2:1 stoichiometric mixtures of purine:pyrimidine Oligomers.
• Figure 18B depicts the temperature dependence of chemical shift for the three resonances observed for the 2:1 mixture: Watson-Crick Gua N1H-Cyt N3 ( ⁇ ); Watson-Crick Thy N3H-Ade N1 (•); and new third strand hydrogen bonded imido protons ( ⁇ ).
• the Watson-Crick assignments were tentative and based on comparison to chemical shifts observed for the d(AG) 8 phosphodiester duplex. These three resonances were easily detected up to 60°C. At 65°C their inten sities decreased dramatically, indicating that the hydrogen bonded complex had, in general, melted.

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