Classifications

• • 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 invention relates to novel oligomer analogs and to oligonucleotide-based diagnostics by binding of the oligomers to single and double- stranded nucleic acids target sequences. More specifically, the invention concerns oligomers containing 8-aminopurine base residues and their triple-helix stabilization properties.
• triple helices offer the possibility of designing sequence- specific DNA binding molecules, which may have important uses as diagnostic tools as well as therapeutic treatments.
• triple helices are used for the extraction and purification of specific nucleotide sequences, control of gene expression, mapping genomic DNA, detection of mutations in homopurine DNA sequences, site-directed mutagenesis, triplex-mediated inhibition of viral DNA integration, nonenzymatic ligation of double-helical DNA and quantitation of polymerase chain reactions (for reviews see references 1-3).
• triplex forming oligonucleotides TFO
• Another problem associated with the use of triplex forming oligonucleotides (TFO) is the presence of interruptions in the homopurine-homopyrimidine tracks.
• TFO triplex forming oligonucleotides
• One of the most successful modifications is to replace natural bases with some modified bases such as 5-methylcytidine, 5-bromouracil, 5-aminouracil, N 4 - spermine-5-methylcytidine, or 5-methyl-2,6(lH,3H)-pyrimidinedione
• the most studied type of triple helix formation is the so-called purine: pyrimidine: pyrimidine motif. ( Figure 1).
• the purine: pyrimidine strands correspond to the target double-stranded DNA sequence (known as the Watson-Crick purine and pyrimidine strands) and the Hoogsteen strand is a pyrimidine strand used for the specific recognition of the double-stranded DNA.
• the base analogues studied for triple helix stabilization are modified pyrimidines located at the Hoogsteen strand, though there are some recent disclosures of purine analogs. For example, see U.S. Patent Nos. 5,739,308; 5,645,985; and 5,594,121.
• inverted polarity is meant that the oligomer contains tandem sequences which have opposite polarity, i.e. one segment or region of sequences having polarity 5'- >3 ⁇ followed by another with polarity 3'->5' or vice versa. This implies that these sequences are joined by linkages which can be thought as a 3 '-3' or a 5 '-5' internucleotide junction.
• Such oligomers named "parallel-stranded DNA” have been synthesized See References 34, 42 and 43.
• oligonucleotides containing 8- aminopurines have been described, but these oligonucleotides can not be directly used for the specific recognition of double-stranded DNA sequences because the modified bases are purines that are in the target sequence and not in the Hoogsteen strand used for the specific recognition of double-stranded DNA.
• Synthetic oligonucleotides probes have been proven very useful in the detection of cloned DNA sequences. When a partial protein sequence is available, a mixture of oligonucleotides presenting all possible DNA sequences can be successfully used as a probe or as PCR or sequencing primers for screening of cloned DNA (or amplification of DNA. See reference 44.
• the mixed probe approach may have two principal drawbacks when the complexity of the mixture is very high.
• a universal base one that could base pair equally well with any of the four natural bases — could resolve these two difficulties.
• a number of compounds have been tested as possible universal bases, with being 2'-deoxyinosine one of the most successfully used. See references 45-50.
• compositions are needed that are capable of specifically selecting target nucleic acid sequences that do not require amplification or that can be used with amplification techniques but provide for more target specific amplification. Additionally, what is needed are compositions and methods that require less use of enzymes.
• compositions and methods for binding to specific target regions of DNA or RNA sequences are compositions and methods for binding to specific target regions of DNA or RNA sequences. Specifically what is needed are triplex structures that are stabile at neutral pHs and modified oligonucletides that can bind to specific sequences in a target nucleic acid to form triple helix structures. Modified oligonucleotides that can be used for synthesis of oligonucleotides in the 5' to 3' direction, reverse of the normal 3' to 5' synthesis direction are also needed. What is also needed are spacer arms that can link oligonucleotides in 5' to 5' orientation or 3' to 3' orientation. Particularly needed are simple and economic methods for the synthesis of such spacer arms and such paired oligonucleotide structures.
• compositions and methods for incorporation of modified nucleic acid bases are also needed. What is particularly needed are methods and compositions comprising a base labeled with active compounds, such as intercalating agents, photoreactive agents and cleavage agents that are attached to the base through a linker arm.
• active compounds such as intercalating agents, photoreactive agents and cleavage agents that are attached to the base through a linker arm.
• the present invention is directed to methods and compositions of nucleic acid structures that are used for detection of specific nucleic acid sequences.
• One of the embodiments of the present invention comprises compositions and methods for the preparation of oligonucleotides carrying modified nucleic acids, such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine, that are connected 3' to 3' or 5' to 5' (head-to-head or tail-to-tail) to a Hoogsteen pyrimidine strand.
• modified nucleic acids such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine
• modified oligonucleotides allow the specific recognition of double-stranded, and single-stranded nucleic acids by binding to the Watson-Crick pyrimidine strand via a triple helix. Additionally, the present invention comprises a universal base oligonucleotide that forms stable base paris with the four natural bases.
• Preferred methods of the present invention comprise binding or capturing a predetermined sequence on DNA or RNA by forming a triple helix that will be stable at neutral pH.
• the stability of the triple helix at neutral pH allows the use of these modified oligonucleotides in conjunction with enzymatic reactions in order to enhance the discriminatory power of the modified oligonucleotides and the direct use of these oligonucleotides under physiological conditions.
• the methods and compositions of the present invention comprise use of hairpin structures to form triplex structures.
• the present invention also provides methods and compositions for detecting a specific nucleic acid target.
• a preferred embodiment comprises methods involving hybridization of hairpin probes that comprise a 5' to 3' purine sequence, followed by a loop sequence, followed by a 3' to 5' pyrimidine sequence that is complementary to the purine sequence.
• These self- annealing probes comprise a region, the loop, that will not hybridize to any of the probe's sequence.
• the loop sequence comprises any sequence that will not hybridize to itself.
• At least one of the hairpin probes can be complexed with a magnetic bead or a molecule that is effective for capture or detection of the structure formed with the hairpin.
• the present invention also comprises compositions and methods for making spacer arms that allow the synthesis of paired parallel stranded oligonucleotides using either 3' phosphoramidite chemistry or 5' phosphoramidite chemistry. Additionally, the oligonucleotide member of the pair an comprise any number of nucleotides.
• the present invention further comprises compositions and methods for synthesis of nucleosides with linker arms for attachment of active compounds.
• the active compounds include, but are not limited to, intercalation agents, photoreactive compounds and agents capable of cleaving nucleic acids.
• the present invention comprises compositions and methods of synthesis and use of pyrimidine nucleosides with linker arms at the N 4 of 2'-deoxycytidine wherein the active compound is a protected fluorescent label and a cleavage agent, such as 5-bromouracil.
• Figure 1 is a drawing of a triple helix with the purine: pyrimidine: pyrimidine motif.
• Figure 2 is a drawing of the base-pairing of triads containing 8- aminopurines.
• Figure 3 is a drawing of a double- or single-stranded nucleic acid (RNA or DNA) binding with oligonucleotide derivatives of the present invention.
• Figure 4 shows a method for making oligonucleotide sequences containing 8-aminopurines using phosphoramidite chemistry.
• Figure 5 is a drawing of the triplex formed using the oligonucleotide composition, B-22A.
• Figure 6 is a drawing of the triplex formed using the oligonucleotide composition, B-22A, and B-22A control where no Hoogstein bonding can occur.
• Figure 7 A and B are drawings of procedures to produce 3 '-3' (a) or
• Figure 8 is a drawing of the synthesis steps of the glycerol-spacer arm (S).
• Figure 9 A and B is a drawing of the synthesis of the 5 '-5' and 3 '-3' attached oligonucleotides.
• Figure 10 shows the synthesis of the glycerol-spacer arm (AS) and attachment to LCAA-CPG using a linker.
• Figure 11A and B show the synthesis of 5'-5' and 3'-3' attached oligonucleotides with different sequences using the AS-glycerol spacer arm.
• Figure 12 shows the synthesis of the 2-nitrophenyl intermediate.
• Figure 13 illustrates a protected fluorescein attached to the 1,2- bis(aminoethoxy)ethane linker arm.
• Figure 14A and B diagram the synthesis of the N 4 -spacer arm- fluorescein labelled-2'-deoxycytidine phosphoramidite.
• Figure 15 shows a method for attachment of the 5-bromouracil to the l,2-bis(aminoethoxy)ethane linker arm.
• Figure 16 A and B details the synthesis of the N 4 -spacer arm-5- bromouracil-2'deoxycytidine phosphoramidite.
• Figure 17A, B and C show the synthesis of the 8-amino-2'- deoxyadenosine monomer.
• Figure 18A and B diagrams the synthesis of the 8-amino-2'- deoxyguanosine reversed monomer.
• Figure 19A and B illustrates the synthesis of the 8-amino-2'- deoxyinosine reversed monomer.
• Figure 20A and B show the synthesis of the 5-methyl-2'-deoxycytidine reversed monomer.
• Figure 21A and B show embodiments of the methods of the present invention using the triplex forming oligonucleotides and detection of the structures formed.
• the present invention is directed to compositions of nucleic acid structures that can be used in molecular biological techniques. Additionally, the present invention is directed to methods of making such nucleic acid structures and to methods of using such nucleic acid structures. These compositions and methods can be used in such molecular biological techniques including, but not limited to, diagnostic tests involving triplex and duplex formations, signal amplification systems, nucleic acid synthesis and others.
• compositions of the present invention comprise parallel- stranded oligomers having at least one 8-aminopurine in the purine strand of general formula: 3 purine strand having 8-aminopurines 5 — linker — 5 pyrimidine strand 3 or 5 purine strand having 8-aminopurines 3 — linker — 3 pyrimidine strand where —linker— comprises any method of coupling nucleotide sequences of opposite polarity.
• the 8-amino purines on the purine strand have the general formula indicated below.
• the present invention also comprises compositions and methods for the preparation of oligonucleotide derivatives constituted by a polypyrimidine part linked head-to-head with a polypurine sequence carrying several 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxan thine.
• Methods of use of these derivatives comprise binding specifically to a predetermined target sequence, preferably a polypyrimidine target sequence, by forming a very stable triple helix that can be observed even at neutral pH.
• the high degree of stabilization obtained by the addition of several 8-aminopurines is especially relevant to the development of new applications based on triple helix formation such as structural studies, DNA-based diagnostic tools, and antigene therapy.
• An embodiment of the present invention comprises oligonucleotide derivatives comprising two parts: a polypyrimidine part connected head-to-head to a complementary purine part carrying one or more 8-aminopurines such as 8- aminoadenine, 8-aminoguanine and 8-aminohypoxanthine.
• a linker molecule is located between both parts in such a way that both parts can form a double stranded structure in parallel sense.
• These oligonucleotide derivatives bind polypyrimidine sequences complementary (in the antiparallel sense) to the purine part by triple helix formation. DNA triple helical structures are normally observed at acidic pH. However, when oligonucleotides carrying 8-aminopurines were used, very stable triple helical structures were observed even at neutral pH. Applications based on triple helix formation will benefit from the use of these oligonucleotide derivatives.
• a polypyrimidine part is connected tail-to-tail to a complementary purine part carrying one or more 8- aminopurines.
• oligonucleotide derivatives carrying 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine connected head-to-head or tail-to-tail to the Hoogsteen pyrimidine strand is taught herein.
• Some embodiments of the sequences of the oligonucleotides of the present invention are shown in Table 1.
• Oligonucleotide derivatives (R-22, R-22A, R-22G, R-22I) contain 22 bases divided in two different parts and connected with a hexaethyleneglycol linker [(EG) 6 ].
• One half corresponds to a polypyrimidine sequence, that will be similar to the target polypyrimidine sequence but with the inverted orientation. This sequence will be the Hoogsteen strand in the triple helix.
• the other half corresponds to a polypurine sequence that will be complementary to the target polypyrimidine in which two purines are substituted by the corresponding 8-aminopurine.
• two adenines are substituted by two 8-aminoguanines (A N ).
• oligonucleotide R22G two guanines are substituted by two 8-aminoguanines (G N ) and in the oligonucleotide R-22I, two guanines are substituted by two 8- aminohypoxanthines (I N ).
• the control oligonucleotide (R-22) contains only the natural bases without modification.
• the second group of oligomers (B-22, B-22A, B-22G) are similar in composition to the previous oligomers but the polypurine and the polypyrimidine parts are connected through their 5' ends with an hexaethyleneglycol linker [(EG)6_
• an oligomer having two 8-aminoguanines and two 8-aminoadenines was prepared (B-22AG) to test whether the stabilizing properties both 8-aminopurines are additive.
• a control oligonucleotide (B-22Acontrol) having the same oligonucleotide sequence in the polypurine part than B-22A but a non-sense polypyrimidine sequence was prepared.
• the oligomer B-22AMMT was prepared to test the influence of interruptions on the stability of the triple helix. In this oligomer there are two adenines substituted by two 8-aminoadenines.
• FIG. 1 In the middle of the purine part there is a pyrimidine (C) and in the corresponding position at the Hoogsteen strand there is a T.
• a third group of oligomers (B-22ALT1, B-22ALT2, B-22ALGA) have the same nucleotide sequence than B-22A but the loop between the polypurine and polypyrimidines parts are made out of nucleotides (-TTTT- and -GGAGG-) instead of the hexaethyleneglycol bridge.
• Figure 3 is a general representation of the binding of the oligonucleotides of the present invention with single or double- stranded nucleic acids.
• a preferred method for synthesizing the oligonucleotides of the present invention comprises use of phosphoramidite chemistry. It is to be understood that the synthesis of the oligonucleotides herein described, or their equivalents, can be synthesized by any methods known to those skilled in the art. See References 34, 41 and 42. In a preferred method, oligonucleotide sequences containing 8-aminopurines were prepared using phosphoramidite chemistry on an automatic DNA synthesizer (see Figure 4). For the preparation of the pyrimidine part, reversed C and T phosphoramidites and reversed C-support (linked to the support through the 5' end) were used.
• the phosphoramidite of 8-aminohypoxanthine was prepared as follows. 8-Amino-2'-deoxyadenosine was deaminated by the action of the enzyme adenosine deaminase (Adenosine aminohydrolase, EC 3.5.4.4), yielding 8-amino-2'- deoxyinosine. Protection of the 8-amino group was performed by addition of the phenoxyacetyl group. See reference 50. The resulting N 8 -phenoxyacetyl derivative was not soluble and was reacted with isobutyryl chloride.
• N 8 - phenoxyacetyl- N 8 -isobutyryl derivative was reacted with dimethoxytrityl chloride in pyridine yielding 8-amino-2'-deoxy-5'-O-dimethoxytrityl-N 8 -isobutyryl-inosine.
• the phenoxyacetyl group was removed due to the lability of this group.
• Direct introduction of the isobutyryl group at N 8 position is possible but yields are lower than the route with the phenoxyacetyl group.
• the phosphoramidite group was introduced in position 3' using chloro-O-2- cyanoethoxy-N,N-diisopropylamino-phosphine as described. See reference 51.
• Methods for detecting formation of triplexes are contemplated by the present invention.
• One of the embodiments of these methods used for the detection of the formation of the triple helix structure comprise the measurement of the triplex fusion temperature, which comprises incubating the samples at increasing temperatures vs. time, and collecting hyperchromicity data.
• Hyperchromicity is the amount by which the UV absorbance of a nucleic acid sample increases when going, for example, from a double stranded structure to a single strand structure as it melts in response to increasing temperature.
• a preferred embodiment of the assay is described below:
• a single stranded template 11 bases long, of constant sequence, in most cases, consisting exclusively of a pyrimidine stretch (WC-1 lmer).
• hairpin probes described above of the same length, where the two strands are joined by a loop consisting of 6 ethylene glycol members or the sequences TTTT or GGAGG; and the purine stretch contain modified bases (i.e., 8- amino A and/or 8-amino G, or 8-amino-I).
• the relative stability of triple helices formed by the R-22 oligonucleotide derivatives and the polypyrimidine target sequence (WC-1 lmer) were measured spectrophotometrically at different pHs (pH 5.5-7.0). In the examples shown herein, one single transition was observed with an hyperchromicity around 25% that was assigned to the melting of the triple helix. Replacement of A and G by 8-aminoadenine (A N ) and 8-aminoguanine (G N ) in triple helix results in a high stabilization of triple helix (10-18°C in the range from pH 5.5 to pH 7.0, see Table 2).
• the relative stability of triple helices formed by the B-22 oligonucleotide derivatives and the polypyrimidine target sequence (WC-1 lmer) were measured.
• the purine and pyrimidine strands are connected by their 5 'ends.
• one single transition was observed with an hyperchromicity around 25% that was assigned to the melting of the triple helix.
• Replacement of A by 8- aminoadenine (AN) and guanine by 8-aminoguanine (GN) in triple helix results in a high stabilization of triple helix (see Table 3) as observed previously.
• the hairpin derivatives comprised in this invention have a preformed structure that will facilitate the formation of triple helices.
• the role of the Hoogsteen strand was further investigated.
• a hairpin probe of the same purine sequence was prepared, but with two 8-aminoadenine substitutions, and with a non-complementary pyrimidine strand.
• This oligonucleotide (named B-22Acontrol) can only form Watson-Crick base pair with the target sequence (WC-1 lmer); but no Hoogsteen bond can be formed ( Figure 6).
• hyperchromicity is the amount by which the UV absorbance of a nucleic acid sample increases when going, for example, from a double stranded structure to a single strand structure as it melts in response to increasing temperature.
• a 10 - 15 % increase is considered to correspond to the (melting) transition from a duplex to a single-stranded structure, in one step.
• a 15 - 25 % increase is considered to correspond to the (melting) transition from a triplex to a single-stranded structure, in one step.
• the transitions observed with the duplexes formed by B22-Acontrol: WC-1 lmer have lower Tm and lower hyperchromicity.
• the hyperchromicity associated with the transition of duplex formed by B-22Acontrol : WC-1 lmer is around 11%, which indicates a transition of duplex to single-stranded.
• the transitions observed with the complex formed by B22A : WC- l lmer have a 22 % hyperchromicity typical of a transition from triplex to single- stranded.
• the difference between the Tm of B-22Acontrol duplex and B-22 A triplex is the gain obtained by the addition of a complementary Hoogsteen strand. At pH 6.0, this difference is 11 °C (1.0 degree per base) and, at pH 5.5, is 16 °C (1.4 degree per base).
• the effect of the salts at pH 6.0 were studied.
• the triplex formed by hairpin having two 8-aminoadenines (A N ) and WC-1 lmer were used as well as the triplex formed by the hairpin having two 8-aminoguanines (G N ) and WC-1 lmer.
• the effect of the concentration of NaCl, MgCl 2 , and spermine on the stability of the triplex was studied. Previous reports on triplexes showed an increased stabilization of triplexes by the addition of magnesium and spermine. See reference 2.
• the effect of these salts on a triplex is shown in the following tables.
• the buffer used was 0.1 M sodium phosphate-citric acid pH 6.0.
• MgCl 2 and spermine the buffer used was 0.1 M sodium phosphate pH 6.0.
• Sodium chloride had a small stabilization effect (from 48.9 °C (no NaCl) to 51 °C (IM NaCl)).
• Low concentrations of magnesium chloride stabilized the triplex, for example the melting temperature of triplex B-22G : WC-1 lmer and B- 22A : WC-1 lmer increases 5 degrees from no MgCl 2 to 10 mM MgCl 2 . Going from 10 mM to 50 mM MgCl 2 the increase on the melting temperature was none or less than one degree.
• adding more magnesium than 50 mM resulted in precipitation of magnesium oxide during the heating.
• the presence of the magnesium is beneficial for the stability of triplex, with 10 mM the optimal concentration.
• the stabilization properties of the 8-aminopurines were additive.
• the addition of the two 8-aminoguanines and two 8-aminoadenines increased the melting temperature by 20 °C, while the increase on the melting temperature induced by two 8-aminoadenine was 6 °C, and the increase induced by two 8-aminoguanines was 14 °C.
• the effect of an interruption on the polypyrimine track of the target was measured.
• a polypyrimidine target with a purine in the middle of the sequence was prepared (s braid-MM : 5 TCTCCTGCTTC 3 ).
• This hairpin carrying two 8-aminoadenines was designed (B-22AMMT: ' AGA N GGA N CGAAG 5' -(EG) 6 - 5' CTTCTTCCTCT 3' ).
• This oligomer has a C opposite to the G of s ⁇ -MM and a T on the Hoogsteen strand opposite to the C:G interruption.
• the choice of T on the Hoogsteen strand is based on previous reports that showed that T is the less destabilizing base in similar situations. Melting curves were performed at pH 6.0, 0.1 M sodium phosphate, 1 M NaCl.
• the introduction of an interruption on the polypyrimidine-polypurine track was studied.
• a guanine was introduced on the polypyrimidine target instead of a cytosine.
• An hairpin with two 8-aminopurines was designed in where a cytosine was located in the purine part opposite to the cytosine.
• a thymine was located in the Hoogsteen pyrimidine part.
• the hairpin B-22AMMT was able to bind to its target (s n -MM) although there is a decrease of 6 degrees in the Tm (44.5 °C B-22AMMT : s n -MM compared with 51 °C B-22A : WC-llmer).
• the binding of the new hairpin to its new target was very selective as judged by the big decrease observed on the Tm of the triplex B-22AMMT with the old target (30.1 °C B-22AMMT : WC-l lmer versus 44.5 °C B-22AMMT : s preference-MM).
• the oligomers of the present invention can be redesigned to cope with the presence of interruptions on the polypurine- polypyrimidine tracts.
• the role of the loop on the stability of the triplex was analysed by preparing derivatives of B-22A with different loops. In addition to the hexaethyleneglycol linker, the nucleotide loops -TTTT- and -GGAGG- were studied. See reference 41.
• Two tetrathymine loops were prepared : one on reversed orientation than the purine strand (B-22ALT1) and the second on the same orientation than the purine strand (B-22ALT2).
• the GGAAA loop was on the same orientation than the purine strand (B-22ALGA).
• Melting curves were performed with the following hairpins and the target WC-l lmer at pH 6.0, 0.1 M sodium phosphate, 1 M NaCl.
• nucleotide loops created more stability of the triplex than the hexaethyleneglycol linker. Best results were obtained with the reversed TTTT linker (hairpin B-22ALT1, an increased on Tm of 6 degrees), followed by the TTTT linker (hairpin B-22ALT2, ⁇ Tm 4 degrees) and the GGAGG linker (hairpin B-22ALGA, ⁇ Tm 3 degrees).
• Binding of hairpins to target were also analysed by gel-shift experiments.
• the target was labelled radioactively with [ ⁇ - 32 P]- ATP and polynucleotide kinase and increasing amounts of the hairpins were added to a solution of the labelled target. After incubation at room temperature for 30 min-1 hr the mixtures were analysed by polyacrylamide gel electrophoresis. The formation of the triplex was followed by the appearance of a radioactive band with less mobility than the band corresponding to the target alone.
• the present invention further comprises compositions and methods for the detection of nucleic acid sequences. These methods include automation procedures for rapid screening and diagnostic applications.
• One embodiment of the present invention is the use of one or more hairpin structures that hybridize to a nucleic acids to form triplex structures.
• the present invention also comprises hybridization of multiple hairpins that, for example, can distort the structure of the nucleic acid and allow for interactions such as intercalating agents, binding of other probes or restriction of the unbound or distorted areas.
• two hai ⁇ in probes are hybridized to one strand of the double-stranded DNA so that the probes flank the target sequence on both sides and form two triplex structures.
• These two triplex structures force the ds DNA to "breathe" open between the triplex structures and allow for binding of other nucleotide structures or probes within this region, if required by a particular method.
• Figure 21 A and B illustrates an embodiment of the triplex formed by addition of self-annealing probes to ds DNA.
• Two hai ⁇ in structures are used to form two triplex regions, and one of the hai ⁇ in structures is used as a capture molecule, in which the loop portion has a peptide attached that can be bound by a specific antibody.
• the capture molecule is a magnetic bead attached to the loop portion of the hai ⁇ in structure and the reporter probe is detected using a monoclonal antibody that is recognized by a second antibody that is labeled using a liposome filled with a fluorophore.
• the entire reacted structure is detected using an antibody that is labeled using a liposome filled with a fluorophore.
• a preferred embodiment comprises methods and compositions involving hybridization of at least one hai ⁇ in probe that comprises a 5' to 3' purine sequence, followed by a loop sequence, followed by a 3' to 5' pyrimidine sequence that is complementary to the purine sequence, as shown in Figure 3.
• the loop sequence comprises any sequence that will not hybridize to itself.
• the loop may be of any sequence or include any number of nucleotides, preferably four thymidine resides, or a six-member ethylene glycol chain.
• the ethylene glycol chain can be derivatized to allow for attachment of different molecules.
• the purine and pyrimidine sequences are approximately eleven nucleotides each.
• the pyrimidine sequence may be synthesized using reverse phosphoramidites.
• at least one of the hai ⁇ in probes is labeled with a peptide, preferably a hexapeptide, comprising a known amino acid sequence.
• Antibodies, that are specific for this amino acid sequence are attached to magnetic beads.
• the binding of the antibody and the peptide binding partner allows for the capture of the DNA target comprising the triplex structures.
• the loop structure of the hai ⁇ in probe is attached directly to a magnetic bead for capture.
• the antibodies used may be monoclonal, polyclonal or antibody fragments. Additionally, other specific binding partner pairs, such as those known in the art, can be used with the present invention.
• a novel detection system comprises use of a single-stranded reporter probe that binds between the two triplex formations, as shown in Figure 21A and B.
• the reporter comprises a nucleic acid sequence that has a peptide attached to it that can be detected by a first antibody.
• a second antibody, specific for the first antibody is attached to a liposome filled with a fluorophore. Upon addition of the complement cascade proteins, the liposome could be lysed and the fluorophore released and thus, detected.
• the detection step may comprise binding of a single-stranded reporter probe having a peptide, preferably a hexapeptide, attached.
• the presence of the peptide is detected using an antibody to the peptide that is complexed with a liposome.
• the liposome is filled with a chosen fluorophore and modified at a very low and limited number of sites with the antibody specific for the peptide of the reporter probe.
• the liposomes of the present invention may comprise different substrates and the outer surfaces may be modified using methods known to those skilled in the art, such as modification methods involving, but not limited to, thiol groups or amino groups.
• Monoclonal antibodies or polyclonal antibodies or antibody fragments could be attached to such liposomes.
• any liposome that can be filled with a detectable agent can be used in the present invention.
• the liposome can be filled with the detectable agent using a variety of methods.
• One method is passive diffusion.
• a liposome of approximately 50 nm in diameter will take up 30 to 40 molecules of carboxy-fluorescein. Larger liposomes, such as those having diameters of approximately 150 nm, can take up more detectable agent molecules.
• With one reporter probe there would be a minimum of 30-fold signal amplification with 50 nm liposomes, over the use of a single detectable molecule attached to a reporter probe.
• the liposomes could be lysed by any method to release the detectable agents.
• mild conditions such as those using ionic detergents could be used to open the liposome channels and release the detectable agent.
• the present invention also comprises multiplexing. Such methods and compositions may be used with heterozygous samples and or SNPs by using reporter probes of different sequence specificity and attached to different peptides. Each peptide/probe would be detected by a different antibody attached to a liposome carrying a detectable agent that is not carried by any other liposome. It is preferred that the detectable agents do not interfere with one another.
• the present invention also comprises compositions and methods of making parallel stranded oligonucleotides. Such methods comprise use of spacer arms and oligonucleotides that may comprise any number of bases. The synthesis of 3 '-3' or 5 '-5' attached oligonucleotides is described herein. The oligonucleotides may have the same or different sequences of nucleotides. Oligonucleotide synthesis is usually carried out from the 3' to the 5' terminus. However, in some cases it is necessary to synthesize oligonucleotides in the opposite sense (5' to 3').
• FIG 8. (S*-symmetrical).
• the glycerol spacer arm was attached to the LCAA- CPG using the oxalyl linker.
• the oxalyl can be replaced by succinyl or sarcosyl spacer arms.
• i) is the reaction of dimethoxytrityl chloride in pyridine
• ii) are the reactions a) oxalyl chloride and 1,2,4-triazole in acetonitrile
• DMAP dimethylaminopyridine
• THF tetrahydrofuran
• the first step of the synthesis consisted of the selective protection of the primary hydroxyl function.
• the protected product was dissolved in acetonitrile/pyridine (2:1) and then added to a previously prepared mixture of oxalyl chloride and 1,2,4-triazole in anhydrous acetonitrile. After 1 hour the solution was transferred to a gastight syringe containing the LCAA-CPG and the reaction was allowed to proceed for 30 minutes. The liquid was ejected from the syringe and the solid support washed successively with acetonitrile, anhydrous methanol (to cap residual oxalyl triazolide groups) and acetonitrile.
• the solid support was treated with an equivolume mixture of tetrahydrofuran (THF) solutions of DMPA and acetic anhydride for 30 minutes. At the end, the support was washed with pyridine, acetonitrile and diethyl ether, air dried and then dried in vacuo.
• THF tetrahydrofuran
• oligonucleotides with identical sequences were prepared on an automatic DNA synthesizer using the glycerol spacer arm (S)-LCAA- CPG and standard or reversed 2-cyanoethyl phosphoramidite chemistry ( Figure 9A & B). Oligonucleotides were synthesized with the last DMT group on to help reverse- phase HPLC-purification. After the assembly of the sequences, the oligonucleotides were cleaved from the support by a treatment of 32% aqueous ammonia at 55 °C for 16 hours. Ammonia solutions were then concentrated to dryness and the products purified using the standard protocols for reverse-phase HPLC.
• glycerol was also used as starting material for the preparation of the spacer arm. Any compound with the structure shown above can be used with good results.
• the first step of the synthesis consisted of the selective protection of one of the primary hydroxyl functions.
• the reaction was carried out in pyridine using an excess of glycerol.
• the monoprotected product was dissolved in anhydrous dichloromethane. 4-Dimethylaminopyridine, triethylamine and laevulinic anhydride were added to the stirred solution with exclusion of moisture, and the reaction mixture was left at room temperature overnight. After the work-up, the desired compounds were purified by column chromatography on silica gel.
• the 1,3-protected glycerol was dissolved in acetonitrile-pyridine (2:1) and then added to a previously prepared mixture of oxalyl chloride and 1,2,4-triazole in anhydrous acetonitrile. After 1 hour the solution was transferred to a gas-tight syringe containing the LCAA-CPG and the reaction is allowed to proceed for 30 minutes. The liquid was ejected from the syringe and the solid support washed successively with acetonitrile, anhydrous methanol (to cap residual oxalyl triazolide groups) and acetonitrile.
• the solid support was treated with an equivolume mixture of tetrahydrofuran (THF) solutions of DMAP and acetic anhydride for 30 minutes.
• THF tetrahydrofuran
• the support was washed with pyridine, acetonitrile, and diethyl ether, air dried and then dried in vacuo.
• 3'-3' and 5'-5' attached oligonucleotides with different sequences can be synthesized on an automatic DNA synthesizer using the glycerol spacer arm (As)- LCAA-CPG and standard or reversed 2-cyanoethyl phosphoramidite chemistry ( Figure 11A and B).
• the dimethoxytrityl-protecting group from the spacer arm was removed, allowing chain extension is that direction to complete the desired sequence. Then, the dimethoxytrityl protecting group of the last monomer added was removed and the resultant hydroxyl group was capped by acetylation to prevent further chain extension from this part.
• the laevulinyl group was cleaved with hydrazine hydrate in pyridine acetic acid.
• the oligonucleotides were cleaved from the support by a treatment of 32% aqueous ammonia at 55°C for 16 hours. Ammonia solutions were then concentrated to dryness and the products purified using the standard protocols for reverse-phase HPLC. Modified Nucleosides.
• the present invention further comprises compositions and methods for the preparation of nucleosides with one or more linkers.
• pyrimidine nucleosides with linker arms at N 4 of 2'-deoxycytidine using the 1,2- bis(aminoethoxy)ethane linker arm are disclosed herein, but any compound with structure shown below can be successfully used. Structures for Preparing Pyrimidine Nucleosides with Linker Arms
• the compounds 1 or 2 can be purchased from commercially available sources.
• the spacer 3 is synthesized by the reaction of l,2-bis(2-chloroethoxy)ethane with a 40% water solution of methylamine in a pressure vessel at 160°C for 7 days.
• the first approach was to synthesize the intermediates needed (see Figure 12).
• the first intermediate synthesized was 3',5'-O- bis(trimethylsilyl)-2'-deoxy-O 4 -(2-nitrophenyl)uridine.
• the first reaction is i), 1,1,1,3,3,3,-hexamethyldisilazane in DMF; ii) a) 0 triethylamine, 2-mesitylenesulphonyl chloride and 4-dimethylaminopyridine in dichloromethane, b) l ,4-diazabicyclo(2.2.2) octane and 2-nitrophenol in dichloromethane. It is contemplated by the present invention that other methods may be used to obtain the intermediates.
• a preferred method of the present invention is the use of protected fluorescein isothiocyanate (supplied by Molecular Probes) in order to avoid modifications of the fluorophore during the oligonucleotide synthesis.
• the compound is designed to produce, on deprotection and isolation of the derived oligonucleotide, the same fluorescein isothiocyanate. See reference 26.
• the reactions of fluorescein isothiocyanate with primary amines are well described in the literature.
• the structure of the protected fluorescein attached to the spacer arm is shown in Figure 13.
• the attachment of 5-bromouracil to the spacer arm is a two step procedure (see Figure 15).
• the attachment of the 5-bromouracil to the 1 ,2- bis(aminoethoxy)ethane linker arm comprised the reactions of i) bromoacetic acid, KOH, and water; and ii) 3,4-dihydro-3-hydroxy-4-oxo-l,2,3-benzotriazine (HOOBt), N,N'-diisopropylcarbodiimide (DIPC), and N-etilmorfoline (NEM) in DMF.
• HOOBt 3,4-dihydro-3-hydroxy-4-oxo-l,2,3-benzotriazine
• DIPC N,N'-diisopropylcarbodiimide
• NEM N-etilmorfoline
• the first reaction was done to produce l-carboxymethyl-5- bromouracil. Due to the structural similarity of 5-bromouracil with thymine, a procedure similar to the one that has been developed to prepare the 1- carboxymethylthymine was used. See reference 33.
• the second step was the attachment of the l-carboxymethyl-5-bromouracil to the 1 ,2-bis(aminoethoxy)ethane linker arm.
• the displacement reaction on the 4 position proceeded rather slowly (overnight) but with very good yields.
• the next steps were the established protocols for the protection of the 5'-hydroxyl function, followed by the phosphitylation reaction the chloro-(2-cyanoethoxy)diisopropylaminophosphine.
• the present invention also comprises compositions and methods of synthesis and use of modified nucleosides. Particularly, the synthesis of reversed phosphoramidites of 5-methyl-2'-deoxycytidine, 8-amino-2'deoxyadenosine, 8- amino-2'-deoxyguanosine and 8-amino-2'-deoxyhyphoxanthine (8-amino-2'- deoxyinosine).
• the 8 -amino-2 ' -deoxy -N 6 ,N 8 -bis-(dimethyl- aminomethyliden)adenosine was prepared as previously described. See references 4,33 and 36.
• the acid labile 9-phenylxanthen-9-yl (Pixyl) group was necessary first to selectively protect the 5 '-hydroxy function. See reference 37.
• the bulky lipophilic tert-butyldiphenylsilyl group carried out this protection. Overnight reaction of the free 3'-hydroxyl group with pixyl chloride pyridine produced the 3'-pixyl protected nucleoside.
• the 8-amino-2'-deoxy-N ⁇ ,N s - bis(dimethyl-aminomethyliden)guanosine was prepared as previously described. See reference 12.
• the acid labile 9-phenylxanthen-9-yl (Pixyl) group on the 3'-hydroxy moiety it is necessary first to selectively protect the 5'-hydroxy function.
• the bulky lipophilic tert-butyldiphenylsilyl group carried ouMhis protection. Overnight reaction of the free 3'-hydroxyl group with pixyl chloride pyridine produced the 3'-pixyl protected nucleoside. This protection can be done alternatively by using dimethoxytrityl chloride, which produces poorer yields.
• the synthesis comprised reactions comprising the following steps, i) Adenosine deaminase in a mixture of dimethylsuphoxide and 0.1M aqueous sodium phosphate (1:3), ii) 1,1,1,3,3,3-Hexamethyldisilazane in DMF, iii) Isobutyryl chloride in pyridine, iv) a) Dioxane/Methanol/25% aqueous ammonia (1:1:2), b) tert-butyldiphenylchlorosilane and imidazole in DMF, v) 9-phenylxanthen- 9-yl (Pixyl) chloride in pyridine, vi) a) tetrabutylammonium fluoride (TBAF) in THF, b) 2-cyanoethoxy-N, N-diisopropylaminochloro phosphine and diisopropylethylamine
• the 8-amino-2'-deoxyinosine was obtained from the treatment of 8-amino-2'deoxyadenosine with adenosine deaminase at 37°C for 72 hours.
• the exocyclic amino protection was then introduced via transient protection procedure to give the N 8 -isobutyryl compound.
• the acid labile 9- phenylxanthen-9-yl (Pixyl) group on the 3'-hydroxy moiety it was necessary first to selectively protect the 5'-hydroxy function.
• the bulky lipophilic tert- butyldiphenylsilyl group carried out the protection.
• the reactions comprise the following steps i) a) 1,1,1,3,3,3- Hexamethyldisilazane in DMF, b) Isobutyryl chloride in pyridine, c) Dioxane/Methanol/25% aqueous ammonia (1:1:2), ii) tert-butyldiphenylchlorosilane and imidazole in DMF, iii) 9-phenylxanthen-9-yl (Pixyl) chloride in pyridine, iv) tetrabutylammonium fluoride (TBAF) in THF, v) 2-cyanoethoxy-N,N- diisopropylaminochloro phosphine and diisopropylethylamine in dichloromethane.
• Oligonucleotides were prepared on an automatic DNA synthesizer using standard and reversed 2-cyanoethyl phosphoramidites and the modified phosphoramidites of the 8-aminopurines prepared as described. See references 4, 5, 10-13.
• the phosphoramidite of the hexaethyleneglycol linker was also obtained from commercial sources.
• Complementary oligonucleotides containing natural bases were also prepared using commercially available chemicals and following standard protocols. After the assembly of the sequences, oligonucleotide-supports were treated with 32% aqueous ammonia at 55°C for 16 hours. Ammonia solutions were concentrated to dryness and the products were purified by reverse-phase HPLC.
• Oligonucleotides were synthesized on 0.2/xmol scale and with the last DMT group at the 5' end (DMT on protocol) to help reverse-phase purification. All purified products presented a major peak which was collected and analyzed by snake venom phosphodiesterase and alkaline phosphatase digestion followed by HPLC analysis of the nucleosides (HPLC conditions B). Yield (OD units at 260 nm after HPLC purification, 0.2 ⁇ mol) were between 6-10 OD. HPLC Conditions
• HPLC solutions were as follows. Solvent A: 5% ACN in 100 mM triethylammonium acetate (pH 6.5) and solvent B: 70% ACN in lOOmM triethylammonium acetate pH 6.5. For analytical runs the following conditions were used. Column: Nucleosil 1210C 18 , 250 x 4 mm, flow rate: 1 ml/min. Conditions A) a 40 min linear gradient from 0 to 75%B. Conditions B) a 20 min linear gradient from
• the nucleoside from Example 2 (1.5 g, 5.6 mmol) was dried by evaporating dry pyridine (10 ml) twice. The resulting residue was dissolved in 20 ml of dry pyridine and treated with phenoxyacetic anhydride (24 mmol, 7.2 g). After 2 hrs of stirring at room temperature water (3 ml) was added to destroy the excess of anhydride and the mixture was concentrated to dryness. A mixture of triethylamine- pyridine-water 20/20/60 was added to deprotect the 3' and 5' -hydroxy functions. After 15 min of stirring at room temperature, the solution was concentrated to dryness. The residue was treated with dichloromethane and the product was not soluble.
• the resulting precipitate was dissolved in 20 ml of pyridine and was treated with trimethylchlorosilane (20 mmol, 2.9 ml). After 25 min of magnetic stirring at room temperature, isobutyryl chloride (16.8 mmol, 1.8 ml) was added and the solution was stirred for 3 hr at room temperature. The mixture was cooled with an ice-bath and water ( 2 ml) was added followed by 2 ml of concentrated ammonia. After 15 min of magnetic stirring at room temperature, the solution was concentrated to dryness.
• Oligonucleotides were prepared on an automatic DNA synthesizer using standard and reversed 2-cyanoethyl phosphoramidites and the modified phosphoramidites of the 8-aminopurines.
• the phosphoramidite of protected 8-amino- 2 '-deoxyinosine was dissolved in dry dichloromethane to make a 0.1 M solution.
• the rest of the phosphoramidites were dissolved in dry acetonitrile (0.1 M solution).
• the phosphoramidite of the hexaethyleneglycol linker was obtained from commercial sources.
• Complementary oligonucleotides containing natural bases were also prepared using commercially available chemicals and following standard protocols.
• oligonucleotide-supports were treated with 32% aqueous ammonia at 55 °C for 16 h except for oligonucleotides having 8- aminoguanine.
• a 0.1 M 2-mercaptoethanol solution in 32% aqueous ammonia was used and the treatment was extended to 24 h at 55 °C.
• Ammonia solutions were concentrated to dryness and the products were purified by reverse- phase HPLC.
• Oligonucleotides were synthesized on 0.2 ⁇ mol scale and with the last DMT group at the 5' end (DMT on protocol) to help reverse-phase purification.
• HPLC purification 0.2 ⁇ mol were between 6-10 OD.
• HPLC conditions HPLC solutions are as follows. Solvent A: 5% ACN in 100 mM triethylammonium acetate
• Double stranded target WC-1 lmer : ' TCTCCTCCTTC 3
• Complementary purine strand 3 AGAGGAGGAAG 5 The target was radioactively labelled at the 5' end by T4 polynucleotide kinase and [ ⁇ - 32 P]-ATP.
• the reaction was performed with 35-50 ⁇ mol of the target oligonucleotide dissolved in 20 ⁇ l of kinase buffer. After incubation at 37°C for 45 min, the solution was heated at 70 °C for 10 min to denature the enzyme and the solution was cooled to room temperature. To the solution 60 ⁇ l of a 50 mM potassium acetate in ethanol were added and the mixture was left at -20 °C for at least 3 hr.
• the mixture was centrifuged at 4 °C for 45 min (14000 ⁇ m) and the supernatant was removed.
• the pellet was washed with 60 ⁇ l of 80% ethanol and centrifuge for 20 min at 4 °C. The supernatant was removed and the pellet was dissolved in 0.2 ml of water.
• the radiolabelled target was incubated with the hairpins of the invention in 0.1 M sodium phosphate / citric acid buffer of pH ranging from 5.5 to 7.0 at room temperature for 30-60 min.
• the hai ⁇ ins were added increasing amounts from 2 to 200 molar equivalents. After incubation the mixtures were analysed by 10- 15% polyacrylamide gel electrophoresis.
• the buffer used on the electrophoresis was the same buffer used during the incubation : 0.1 M sodium phosphate / citric acid buffer of pH ranging from 5.5 to 7.0.
• the formation of the triplex was followed by the appearance of a radioactive band with less mobility than the band corresponding to the target alone.
• the sequences of the hai ⁇ ins are shown in Table 1.
• a preferred method of use of the present invention comprises the following steps. 1. Mildly heat the sample DNA at 50 -60° C for approximately 10 minutes.

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