Patent Application
20060074041
The present invention relates generally to antisense and antigene oligonucleotides and their use as probes as well as diagnostic and therapeutic agents, and more particularly to antisense and antigene oligonucleotides which are capable of topologically linking to target nucleic acid molecules so as to impart tight binding characteristics and, in turn, improved translation and transcription inhibitory properties. The present invention also relates to novel methods for the platination of oligonucleotides to improve their antisense and triplex-forming properties and to allow those oligonucleotides to bind to double-stranded DNA through an antisense mechanism.
Antisense approaches to the therapeutic modulation of gene expression have been shown to be effective both in cultured cells and in whole animals (Crooke, Antisense Nucleic Acid Drug Dev. 8:115-122 (1998), Matteucci and Wagner, Nature 384:20-21 (1996) and Mesmaeker et al., Acc. Chem. Res. 28:366-374 (1995)). However, the standard antisense method, involving delivery of oligonucleotides modified for nuclease resistance, has several difficulties: Toxicity of phosphorothioate and other derivaties is a concern, cell-specific delivery is difficult, and levels with target cells can only be controlled by frequency of injection. In addition, automated synthetic methods for production of antisense oligonucleotides remain expensive, and may prevent the use of clinically successful antisense therapeutics for some indications. A stubborn problem with the introduction of exogenous oligonucleotides is that in many cells they are taken up by endocytosis and confined to endosomes rather than reaching the nucleus. This compartmentalization problem can be circumvented by transcription of antisense genes in situ to generate antisense RNAs or, often, by the delivery of oligonucleotides using cationic lipids. The in situ approach has the added advantages of avoiding unnatural, toxic DNA derivatives and permitting continuous exposure to an antisense molecule or a ribozyme if the introduced DNA is stably expressed. Moreover, with appropriate regulated promoters, delivery can be regulated by external signals. However, antisense RNA cannot take advantage of RNase H cleavage to achieve effective targeting of coding regions. Modifications at the ribose 2′ position which improve effectiveness also prevent RNase H activity. Without activity by RNase H, sense-antisense complexes within coding regions are disrupted by the passage of ribosomes during translation. Double-stranded RNA modification enzymes can also disrupt sense-antisense complexes. Thus, at present it is not possible to achieve blockage of translation in coding regions by hybridization with antisense RNAs. Most, if not all, antisense leads being developed for (or presently in) clinical trials bind mRNA in noncoding regions.
In light of the above, there is a significant need for novel antisense and antigene compositions and methods for using those compositions which effectively provide for the down regulation of protein expression both in vitro and in vivo without many of the limitations inherent in the standard antisense approaches discussed above. Specifically, there is a need for novel antisense and antigene therapeutics which are capable of resisting dissociation after binding to a target RNA molecule through various forms of topological linkage, thereby effectively inhibiting transcription and/or translation from the target.
The present invention is directed to improved antisense and antigene oligonucleotide compositions and methods for down-regulating gene expression in cells using novel antisense and antigene oligonucleotides that are capable of topologically linking to target nucleic acid molecules, thereby imparting tight binding properties and the ability to resist dissociation from the target. More particularly, the present invention is directed to a new generation of antisense and antigene agents for the specific control of gene expression. These agents bind to RNA and/or DNA target molecules not merely by the strength of Watson-Crick pairing (as do standard antisense agents), but employ additional features that “lock” the antisense or antigene molecule onto the target nucleic acid, thereby making it highly resistant to dissociation promoted by helicases, ribosomes or modifying enzymes. We herein demonstrate success in achieving extremely tight binding and have identified mechanisms responsible for this tight binding. Moreover, we herein establish that this method is very effective in blocking ribosome scanning in cell-free translation systems as well as in intact cells. This approach of using nucleic acid structural considerations to topologically “padlock” the antisense or antigene and target molecules together is a significant advance, for antisense and antigene therapy in general, for gene function analysis and target validation and for gene therapy, as a means for the controlled and cell-specific delivery of antisense and antigene molecules, in particular. Methods employing these antisense oligonucleotides both in vitro and in vivo as well as kits comprising them are also provided. Methods for the platination of oligonucleotides, for example, to improve their antisense and triplex-forming properties are also provided. In addition, we provide methods for detecting and amplification of nucleic acids based on mechanistic features of some of these antisense constructs.
FIGS. 1A-H. Exemplary Schemes for Employing Antisense Oligonucleotides Which Become Topologically Linked to the Target Molecule. A. Presented is a general scheme showing a nucleic acid molecule topologically linked to a single-stranded target. The ball represents any of various ways for the ends of this molecule to interact following hybridization with the target with creation of at least one turn of helical interwinding. The third-strand interaction to form a triplex is optional. In one embodiment, the ball comprises a hairpin ribozyme moiety (see
FIGS. 2A-F. Schematic Representation of the Structures of Various ATR 1 Antisense RNA Species and Hairpin Ribozyme Structure. A. Presented is the sequence of the primary transcript pre-ATR 1 (R2) showing its functional domains including the proximal (P5 and P8) and distal (D8 and D9) substrate portions of the hairpin ribozyme (HPR) relative to the catalytic core thereof (E48) (where the numbers refer to the length of the segment in nucleotides), and the antisense (A) and triplex (T) forming regions. Also presented are the fragments generated as products of either R2 self-processing (R3a, R3b and R4) or transcription using a shorter template lacking the catalytic hairpin ribozyme domain (AT). The terminal groups are as shown: “ppp”, 5′-triphosphate; “OH”, 5′ or 3′ hydroxyl; and “>p”, 2′,3′-cyclic phosphate. B. Presented is the putative secondary structure of the complex formed between the murine TNFα mRNA target and the “folded” form of the ATR 1 antisense RNA (R1 or R4). C. Presented is the putative secondary structure of the complex formed between the TNFα mRNA target and the AT antisense RNA, containing only the antisense and triplex forming regions but lacking the hairpin ribozyme. D. Presented is the secondary structure and sequence of the essential core of HPR, showing domains referred to in the text. E. Autoradiograph of a 6% denaturing polyacrylamide gel showing RNA species produced by self-processing of primary HPR1 transcript. Lane 1, 5S RNA marker, lane 2, internally 32P-labeled RNAs showing all species, lane 3, RNAs labeled at their 3′ termini by ligation to 5′-[32P]pCp using T4 ligase. R2 and R3 are unprocessed and partially processed primary transcripts, whereas R1 is circular HPR and R4 is linear HPR. F. 6% denaturing polyacrylamide gel showing RNA species produced by self-processing of primary HPR1 transcript. Lane 1, gel purified circle, lane 2, gel purified linear, lanes 3 and 4, circle (R1) and linear HPR(R4), showing self-cleavage and self-ligation, respectively, after incubating with Mg2+-containing buffer for 1 hour at 37° C.
FIGS. 4A-B. TNFα-Luciferase Fusion Targets and Inhibition of Expression by ATR Antisense RNAs. A. Presented is a schematic diagram and the nucleotide sequence of a DNA template for the TNFα-luciferase fusion gene. B. Presented is the inhibition of luciferase expression detected by a luminescence assay as a result of the pre-hybridization of the luciferase-coding mRNA (PS or PTS) with either AT, ATR 1, or a control RNA lacking the antisense sequence folowed by translation using a rabbit reticulocyte system.
FIGS. 6A-D. Phase Contrast and Fluorescence Microscopy of Hairpin Ribozyme Minimonomer Constructs in Murine Macrophages Following Intraperitoneal Administration. A. Presented is the phase contrast image of responsive murine peritoneal macrophages 24 hours after administration of 10 μg of fluorescein-12-UTP m101 complexed at a 3:1 charge ratio with Lipofectamine in 1 ml of Hanks Balanced Salt Solution (HBSS). B. Present is the fluorescent image of A, showing fluorescein signal in the majority of macrophages, but not lymphocytes. C. Presented is the phase contrast image of responsive murine peritoneal macrophages 24 hours after administration of 10 μg of fluorescein-12-UTP m101 without Lipofectamine. D. Presented is the fluorescent image of C.
FIGS. 12A-D. Kinetics of Hybridization and Strong Complex Formation for ATR 1, ATR 16a, ATR 16b and ALR 229 Constructs with TNFα RNA. FIGS. 12A-D show the kinetics of hybridization and strong complex formation for the ATR 1, ATR 16a, ATR 16b and ALR 229 contructs with TNFα RNA target. cc, complementary complex; sc, strong complex.
FIGS. 17A-C. Mechanisms of oligonucleotide platination and structure of platinum adducts. A, diethylenetriamine chelating a chloroplatinate group tethered to a phosphorothioate residue at the sulfur atom. B, diethylenetriamine chelating a chloroplatinate group tethered to the N7 atom of either one or two neighboring guanine residues. C, oligonucleotides labeled with diethylenetriamineplatinum(II) group at different sites.
FIGS. 18A-E. Platination patterns for oligonucleotides to stabilize complementary complexes with single-stranded nucleic acid targets. a, Cationic platinum groups introduced into internal regions of antisense oligonucleotides through the modification of either sulfur atoms of internucleotide phosphorothioates or N7-positions of purine bases. b-e, Cationic platinum groups attached to antisense oligonucleotide constructs through non-hybridizing sequences or linkers by modification of sulfur atoms of internucleotide (and/or terminal) phosphorothioates, or thio-pyrimidines, or thio-containing peptides or other organic oligomers. •—Represents platinum group attached to oligonucleotide prior to hybridization. Each pair of diagrams shows nucleic acid complexes made by both unmodified and platinated oligonucleotides.
FIGS. 19A-E. Platination patterns for oligonucleotides to stabilize triple-helical complexes with either DNA duplexes, or hairpin and single-stranded RNAs. a, “Classic” triplexes with either homopurine or homopyrimidine tfos. b, alternate strand triplexes with oligonucleotides containing both homopurine and homopyrimidine triplex-forming domains. c, triplexes formed by hybrid oligonucleotides containing two triplex-forming domains connected by a non-hybridizing linker. d, oligonucleotides forming both a duplex and a triplex with a target structure containing an internal loop. e, triplex “clamps” featuring hairpin-like structure. •—represents a platinum group attached to oligonucleotide prior to hybridization. Each pair of diagrams shows nucleic acid complexes made by both unmodified and platinated oligonucleotides.
FIGS. 30A-H. Exemplary Schemes for Stabilization of Topological Linkage by Various Means. In A-G, the Watson-Crick non-covalent base pairing is sufficiently weak that the binding of the padlocking or “clasp” molecule is required for stability. In
The present invention is directed to improved antisense and antigene oligonucleotide compositions and methods for down-regulating the expression of various proteins in cells using novel antisense and antigene oligonucleotides which are capable of resisting dissociation from target nucleic acid molecules. More particularly, the present invention is directed to novel antisense and antigene molecules and methods of their use, wherein the novel antisense and antigene molecules are capable of tightly binding to a target nucleic acid not only through standard Watson-Crick pairing (as do standard antisense agents), but also employ additional features that topologically “lock” the antisense or antigene molecule onto the target nucleic acid molecule, thereby making it highly resistant to dissociation promoted by helicases, ribosomes or modifying enzymes and, in turn, imparting improved translation inhibitory properties.
By “topologically linked” is meant that the antisense or antigene oligonucleotide circularizes around the target molecule. For example, if an initially linear antisense or antigene molecule binds to an mRNA target, wraps around it, and then circularizes, it would be very difficult to displace. Unless an endonucleolytic cleavage event occurs in the circular molecule, hydrogen bonds between the two molecules would have to be simultaneously broken, then the mRNA would have to thread its way out of the circle. Although this is theoretically possible, secondary structure in the mRNA would make it kinetically extremely slow. Such molecules are referred to herein as “topologically linked” to the RNA target.
There are various mechanisms by which the novel antisense and antigene molecules of the present invention may become topologically linked to a target nucleic acid molecule. For example, one may employ an antisense RNA to which a catalytic RNA molecule is linked, either through a natural nucleic acid bond or a linking structure. The catalytic RNA molecule is capable of causing the 5′ and 3′ ends of the antisense oligonucleotide to covalently or non-covalently interact with one another, thereby effectively “topologically” linking the antisense molecule to the target nucleic acid. In one embodiment, the catalytic RNA which finds use in the antisense or antigene molecules of the present invention is the hairpin ribozyme which is derived from the minus strand of the satellite RNA associated with tobacco ringspot virus (Buzayan et al., Nature 323:349-353 (1986a), Feldstein et al., Gene 82:51-63 (1989) and Hampel and Tritz, Biochemistry 28:49294933 (1989)). The catalytic domain of the hairpin ribozyme has a compact and stable structure and is capable of autocatalytically cleaving and ligating at a specific site to interconvert between a covalently closed circle and a non-covalently closed form which possesses a 5′OH group and a 2′,3′-cyclophosphate terminus (
To further stabilize the antisense or antigene molecule/target complex, one can also introduce a triplex-forming region into the antisense or antigene molecule. A triplex-forming region is a nucleic acid sequence which is incorporated into the antisense or antigene molecule and which functions to form a triplex with the duplex that is created between the complementary sequences of the antisense or antigene molecule and its target. While more detail regarding triplex formation and the sequences required therefor is presented below, it is evident to those skilled in the art that the ability to employ triplex-forming sequences will depend upon the sequence of the target nucleic acid and the corresponding antisense or antigene molecule.
Another mechanism by which the novel antisense or antigene molecules of the present invention may be effectively topologically linked to the target molecule is by incorporating a sequence therein which forms a structure upon specifically binding to the target molecule, wherein the formed structure is subsequently bound by a “locking molecule” which essentially serves as a “clasp” that does not allow the antisense or antigene oligonucleotide to be easy displaced from the target molecule. The sequences which function to serve as the binding site for the “locking molecule” are generally placed at the ends of the antisense or antigene molecule, so as to allow them to interact when the molecule is bound to the target. When these ends interact to form the locking molecule binding site, they often form a structure which is sufficiently weak so as to be unstable. However, when this structure is actually bound by the locking molecule, the structure is stabilized and remains tightly bound to the target molecule. As described above, the locking molecule can be virtually anything which binds to a nucleic acid structure in a sequence- or structure-specific manner including, for example, proteins, nucleic acids, metal ions (either by themselves or complexed to other components such as nucleic acids, and the like), organic or inorganic molecules, drugs, and the like. FIGS. 30A-H provides a schematic illustration of some of these mechanisms. Additional detail for this method will be provided below.
The above described antisense and antigene molecules form a tightly bound complex with the target molecule due to their ability to be topologically linked or circularly fixed around the target. In this regard, there are many advantages for circularly linking an antisense or antigene polynucleotide to a target nucleic acid molecule. For example, circular RNA molecules are generally more stable in the cellular environment than are linear RNA molecules. Linear antisense RNAs, either as in situ transcripts from inserted genes or as ribozymes injected into the body, are particularly susceptible to degradation by nucleases in the cell as well as in extracellular fluids such as blood. However, circularization prevents damage from the most prominent nucleases, which are exonucleases. For example, in vitro studies have demonstrated that the half-life of covalently closed circular oligonucleotides in serum is at least 100 times higher than that for linear oligomers (Nilsson et al., Science 265:2085-2088 (1994)). Moreover, in nuclear and cytoplasmic extracts from HeLa cells, circular ribozyme molecules have better resistance to nuclease degradation than do linear forms of the ribozyme (Puttaraju et al., Nucl. Acids Res. 21:4253-4258 (1993)).
Another advantage to circularly linking an antisense or antigene oligonucleotide to a target nucleic acid molecule is improved strength and specificity of binding as compared to that obtained with linear antisense or antigene oligonucleotides. It has been shown that antisense circles as small as 30 to 40 nucleotides can form regular 15-18 bp duplexes with target sequences in mRNAs (Dolinnaya et al., Nucl. Acids Res. 21:5403-5407 (1993)). In the case of DNA, circular oligodeoxyribonucleotides have been shown to bind effectively to single-stranded homopurine or homopyrimidine nucleic acids by triplex formation (Kool, J. Amer. Chem. Soc. 113:6265-6266 (1991)). They show higher sequence selectivity and less tolerance of mismatches with target sequences than do ordinary linear oligomers (Kool (1991), supra, Prakash and Kool, J. Chem. Soc. Chem. Commun. 1161-1163 (1991), Prakash and Kool, J. Am. Chem. Soc. 114:3523-3527 (1992), Wang and Kool, Nucl. Acid. Res. 22:2326-2333 (1994)). A comparison of circular RNA and DNA oligonucleotides of the same sequence showed that RNA circles bind single-stranded RNAs with considerably higher affinity than do DNA circles, even without topological linkage of the oligonucleotides to the targets as described herein (Wang and Kool (1994), supra).
Small RNA molecules are also advantageous as antisense and antigene agents because they minimize possibilities for folding into alternate conformations that can interfere with target recognition (Forster and Symons, Cell 50:9-16 (1987), Hélène and Toulmé, Biochim. Biophys. Acta 1049:99-125 (19900 and Dolinnaya et al. (1993), supra). However, for in situ expression, additional sequences are needed for high-levels or cell-type-specific expression. If a circular RNA is to be the active antisense or antigene agent for the reasons described above, the best way to lessen conformational problems is to autocatalytically excise from the final circle any sequences that are irrelevant for target binding.
We consider herein that the hairpin ribozyme is appropriate for topologically linking an antisense or antigene oligonucleotide to a target nucleic acid because of the compact and stable structure of its catalytic domain (Feldstein and Bruening, Nucl. Acids Res. 21:1991-1998 (1993), Anderson et al., Nucl. Acids. Res. 22:1096-1100 (1994) and Butcher and Burke, J. Mol. Biol. 244:52-63 (1994)) and its high catalytic activity in experiments both in vitro and in vivo (Yu et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993), Chowrira et al., J. Biol. Chem. 268:25856-25864 (1994)). The hairpin ribozyme is derived from the minus strand of the satellite RNA associated with tobacco ringspot virus. The precursor RNA is synthesized in an infected cell as a multimer that self-cleaves to a monomeric unit. The monomeric form freely interconverts between a covalently closed circle and a noncovalently closed form containing a 5′-hydroxyl and a 2′,3′-cyclophosphate terminus. Autocatalytic cleavage and ligation occurs at a specific site (see
The hairpin ribozyme can be separated into catalytic, substrate, and substrate-binding moieties. Mutagenesis, deletion analysis, chemical structure mapping, and in vitro selection experiments have identified a minimal 48-nt sequence, herein termed E48 or the minimonomer, and secondary structure requirements essential for the hairpin ribozyme autocatalytic function (Hampel et al., Nucl. Acid Res. 18:299-304 (1990), Feldstein and Bruening (1993), supra, Anderson et al. (1994), supra and Butcher and Burke (1994), supra) (see
Triplexes: The most common pairing motif for nucleic acid triplexes consists of a pyrimidine third strand pairing with a Watson-Crick duplex, where thymine recognizes A:T base pairs, protonated cytosines recognize G:C base pairs and the third strand is parallel to the purine strand of the duplex. We have recently demonstrated (Jayasena and Johnston, Biochemistry 31:320-327 (1992a), Jayasena and Johnston, Nucl. Acids Res. 20:5279-5288 (1992b) and Jayasena and Johnston, Biochemistry 32:2800-2807 (1993a)) that this motif can be combined with a second, purine third-strand motif to permit triplex formation at sequences other than pure homopurine-homopyrimidine blocks. This capability will permit the antisense- or antigene-triplex approach proposed herein to be applicable to a greater variety of target sequences than would otherwise have been possible.
Triplex formation may require protonation of cytosines in the third strand. Because the pKA of cytosine is shifted substantially toward neutral in a triplex, the triplexes proposed are expected to be stable under physiological conditions. However, if triplex stability is limiting, constructs will be synthesized with 5-methyl cytosine in place of cytosine at the third strand positions, which shifts the pK to higher values.
Any gene in which at least a portion of the coding and/or non-coding sequence is known or readily obtainable and which would benefit from a lower expression thereof will serve as targets for the novel antisense and antigene molecules of the present invention. As will be apparent from the present disclosure, the antisense and antigene oligonucleotides of the present invention can be routinely adapted to bind to and become topologically linked to virtually any target nucleic acid molecule of interest. As such, the presently described antisense molecules represent a major step forward in the field of antisense therapeutics.
Delivery into Cells: Previous studies have demonstrated that catalytic RNA can be delivered to cells utilizing cationic lipids, such as N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) (Sioud et al., J. Mol. Biol. 242:831-835 (1991)). Other investigators (Zhu et al., Science 261:209-211 (1993)) have used a 1:1 ratio of DOTMA with dioleoylphosphatidylethanolamine (DOPE) to achieve systemic expression of plasmid DNA following intravenous injection of plasmid DNA:DOTMA:DOPE complex into mice. Other cationic lipid formulations have become commercially available (e.g. DOSPA:DOPE, DOTAP, DMRIE:cholesterol, DDAB:DOPE, and others) and offer improved expression of DNA. A 1:1 mixture of dioctadecylamidoglycylspermine (DOGS):DOPE is effective to introduce ribozyme constructs into cells both in vivo and in vivo (see, e.g., Kisich and Erickson, J. Leukocyte Biol. Suppl. 2:70 (abstract) (1991a) and Kisich and Erickson, FASEB J. 4:1860 (abstract) (199 lb)). As presented below, 1:1 DOSPA:DOPE (Lipofectamine, Life Technologies, Inc.) is shown to be effective for introducing padlock RNAs into mouse macrophages in vitro and in vivo.
The antisense and antigene molecules of the present invention will find use for reducing or inhibiting expression of a target gene both in vitro and in vivo. For administration, the antisense and antigene molecules may be directly administered by various techniques which are known in the art including transfection, transformation, infection, and the like. Additionally, expression constructs may be employed to provide an expression template (inducible or not) that can provide a continuous source of the antisense or antigene molecule of interest to the cell(s). As such, gene therapy methods are encompassed within the present invention. Vehicles for introducing and inducing expression of introduced nucleic acids are well known in the art and may be readily employed herein.
We also herein describe novel methods for platination of antisense or antigene oligonucleotides and triplex forming oligonucleotides (TFO) through either selective modification of phosphorothioate (POS) linkages in homopyrimidine sequences or guanine residues in purine-rich oligonucleotides.
Effect of metals and polyamines on triplex formation. The fact that triple-stranded nucleic acid structures are usually less stable than related duplexes, particularly if the third strand has backbone modifications such as phosphorothioate substitutions, is a factor limiting the use of the TFOs as antigene agents (Wilson et al., 1993; Lacoste et al., 1997). The lower stability of nucleic acid triple-helical interactions is caused, at least in part, by the added electrostatic repulsion of the third chain relative to duplexes. Moreover, most triplexes employing pyrimidine-rich TFOs are not stable at physiological pH (due to need for cytosine protonation), and physiological concentrations of K+ may hamper triplex formation with purine-rich TFO's (Thuong and Helene, 1993). The negative effect of K+ on a purine-purine-pyrimidine triplex formation is presumably due to self-association of the oligonucleotides in competitive structures such as parallel duplexes (GA-rich TFOs) and/or tetraplexes (GGGG-containing TFOs) (Musso and Van Dyke, 1995; Olivas and Maher, 1995a; Lacoste et al., 1997). Some mono-, di- and multivalent metal cations (Malkov et al. 1993; Thuong and Helene, 1993; Kazakov, 1996; Ellouze et al., 1997), as well as cationic polyamines (Thomas and Thomas, 1993; Musso and Van Dyke, 1995; Pallan and Ganesh, 1996) favor triplex (both pyrimidine- and purine-rich) formation.
However, the inhibitory effect of K+ can be completely overcome or reversed by physiological concentrations of such favorable cofactors as Mg2+, spermine4+ or spermidine3+ (Musso and Van Dyke, 1995; Olivas and Maher, 1995a). Approaches to destabilizing aggregates of purine-rich TFOs under physiological conditions would aid their biological applications (Olivas and Maher, 1995a; Svinarchuk et al., 1996). A remarkable solution for this problem has been accomplished by using 6-thioguanine substituted for guanine, presumably because the increased radius and decreased H-bonding ability of sulfur in the C6-position destabilize potential guanine tetraplexes (Olivas and Maher, 1995b). We propose that platination of the guanine N7-position (see
Attachment of functional groups to triplex-forming and antisense oligonucleotides. The enhancement of triplex stability by cations can be further exploited by their conjugation with the TFOs. For example, the attachment of different cationic peptides (Tung et al., 1996) and polyamines such as spermine (Tung et al., 1993) to the 5′-end of the homopyrimidine oligonucleotides boosts the stability of triplexes while having no affect on the stability of the underlying double helix.
Here, we propose the attachment of conjugates of platinum and polyamine cations to TFOs. Currently, there is no information available about the effect on triplex stability of the of platinum complexes tethered to TFOs. However, it is reasonable to expect that appropriate “platination” of oligonucleotides will not compromise their ability to form triplexes, and might even have positive effects. For example, some other metal complexes, [Fe2+-EDTA] and [Cu+-phenanthroline], attached to oligonucleotides at either their termini, bases or sugar moieties, have been successfully used as chemical probes of triplex structures (Moser and Dervan, 1987; Francois et al., 1989; Beal and Dervan, 1992; Jayasena and Johnston, 1992; Thuong and Helene, 1993; Shimizu et al., 1994; Tsukahara et al., 1996).
Covalent attachment of other functional groups to the oligonucleotides has been employed to alter their affinity for both double- and single-stranded complimentary nucleic acids as well as for introducing reporter groups for structure analysis, providing non-radioactive labels, or preparing synthetic “exonucleases” (see for review: Helene 1993; Thuong and Helene 1993; Plum and Pilch, 1995; O'Donnel and McLaughlin, 1996; Haner and Hall, 1997). Many chemical and biochemical approaches can be used for introducing additional functional groups into commercially available, deprotected oligonucleotides or their derivatives (see for review: Thuong and Helene, 1993; O'Donnel and McLaughlin, 1996). Among these, the functionalization of termini and internucleotide phosphorothioate groups offers a number of advantages over the modification of nucleoside residues (Chu and Orgel, 1994; Fidanza et al., 1994; O'Donnel and McLaughlin, 1996). One advantage is that the attachment of a functional group or label at such sites should not drastically alter the stability of nucleic acid complexes. The alkylation of sulfur in terminal phosphorothioates by substituted alkyl halides was extensively used for functionalization or/and labeling of both antisense and triplex-forming oligonucleotides (Thuong and Helene, 1993; Chu and Orgel, 1994; Shimizu et al., 1994; Tavitian et al., 1998).
The internucleotide phosphorothioate diesters are not as nucleophilic as terminal phosphorothioate esters or alkyl thiols (O'Donnel and McLaughlin, 1996). However, reactive groups such as haloacetamides, azirinylsulfonamides, γ-bromo-α,β-unsaturated carbonyl, and monobromobimane (O'Donnel and McLaughlin, 1996), as well as divalent mercury and platinum (see below) can be used to modify the thioester, forming covalent adducts which are stable under neutral and acidic conditions but can undergo hydrolysis at high pH. An important feature of this approach is the precise placement of the functional group according to the position of the individual phosphorothioate in the synthetic oligonucleotide (Ozaki and McLaughlin, 1992). Moreover, the modification of every internucleotide POS allows the incorporation of multiple groups; ideally one for each POS residue (Conway et al., 1989; Hodges et al., 1989; Conway and McLaughlin, 1991). The nucleophilicity of sulfur in phosphorothioate oligonucleotides has also been used to conjugate them with platinum reagents (see below).
Specific platinum labeling of phosphorothioate nucleic acids. Platinum (and mercury) compounds can specifically bind to sulfur atoms either occurring naturally (e.g., in tRNA molecules) or synthetically incorporated in nucleic acids (Pal et al., 1972; Jones et al., 1973; Scheit and Faerber, 1973; Strothkamp and Lippard, 1976; Strothkamp et al., 1978; Szalda et al., 1979; Chu and Orgel, 1989, 1990, 1991, 1992; Elmroth and Lippard, 1994; Slavin et al., 1994). The resulting modified nucleic acids are potentially useful for X-ray crystallography, electron microscopy or other applications requiring heavy metal labeling (Strothkamp and Lippard, 1976; Lippard, 1978; Strothkamp et al., 1978; Szalda et al., 1979), as well as antisense and antigene probes (Chu and Orgel, 1989, 1990, 1991, 1992).
However, little quantitative information is available about the reactivity of phosphorothioates in nucleic acids toward platinum reagents, and only a few kinds of such reagents have been studied so far. One of them is [(terpy)PtIIx]n+(
Because of the polyelectrolyte effect (Gueron and Weibuch, 1981; Elmroth and Lippard, 1994), cations and H-bond donors are selectively attracted to the negatively charged surface of the oligo- and polynucleotides. Consequently, high local concentration in the vicinity of the polymer molecules, aggregation on the polymer surface and high mobility along the polyanion backbone may significantly enhance the reactivity of attracted cationic reagents. Conversely, the reactivity of anionic reagents towards polyanions should be significantly less in comparison to reactivity with monomers or neutrally charged polymers, and should be enhanced for positively charged polymers. Elmroth and Lippard (1994) showed that such polymer surface effects provide approximately 25-fold higher rate for formation of the Pt—S linkage with d(TTTTTTTSTTTTTTT) in comparison to platination of the dinucleoside monophosphate d(TST) by cis-[Pt(NH3)(NH2C6H11)Cl(H2O)]+ (an analog of the reactive form of the well known anticancer agent cis-[Pt(NH3)2Cl2] (
Orgel and co-workers used oligonucleotides containing phosphorothioate and cystamine groups for specific crosslinking of DNA/RNA duplexes (Chu and Orgel, 1989, 1990a; 1990b), DNA triplexes (Gruff and Orgel, 1991) and DNA-protein complexes (Chu and Orgel, 1992) by different platinum reagents. The crosslinking experiments were performed in buffer solutions containing 30-50 mM NaClO4, 1-7 mM Na-phosphate (pH 7-7.4) and 0.025-0.1 mM EDTA at room temperature overnight in the presence of a large excess of platinum reagent (1-5 μM) over oligonucleotide derivatives (18-72 nM) (Chu and Orgel, 1989; 1990a). The authors could not determine the yield and nature of platinum complexes with POS oligonucleotides because these complexes did not give sharp bands on electrophoresis but they noticed that the crosslinking occurred much more efficiently with phosphorothioate oligonucleotides than with normal all-phosphodiester oligonucleotides (Chu and Orgel, 1989, 1992). However, they noticed higher reactivity of positively or neutrally charged cis-/trans-[(NH3)2PtCl2] (and products of their hydrolysis) than negatively charged K2[PtCl4] (Chu and Orgel, 1990a). Chu and Orgel (1990a) have also discussed the possibility of intramolecular crosslinking into phosphorothioate oligonucleotides by K2[PtCl4] having four reactive Pt—Cl coordinates, noticing that such crosslinking products (if they were formed) were not stable and underwent further chemical transformation (Chu and Orgel, 1990a). Diethylenetriamine Catalyzes Platination of Oligonucleotides. In aqueous solutions, [PtCl4]2− (
The major advantages of this new synthetic method are related to the labeling of oligonucleotide probes by radioactive molecules. This is a one-tube reaction which can be performed in any biochemical lab. No special equipment or skills for multiple steps, radioactive synthesis of platinum compounds and product isolation procedures are required (see for comparison Hoeschele et al., 1980; Anand and Wolf, 1992; Azure et al., 1992). Because of the comparatively fast decay of the radioactive platinum isotopes 193m Pt and 195mPt [t1/2 about 4 days (Stepanek et al., 1996)], the short overall time of the oligonucleotide labeling (a few hours, including purification) is very important. In contrast, long (from overnight to several days) multi-step synthetic procedures are required for preparation of alternative platinum reagents such as [(dien)PtCl]Cl (
The fact that phosphorothioate oligonucleotides have already been subjected through extensive biological tests would make it easy to repeat similar studies using the platinated analogs.
Preparation and Purification Methods of Phosphorothioates. A terminal phosphorothioate can be easily attached to the 5′-end of both RNA and DNA of unmodified oligonucleotides by polynucleotide kinase and ATPyS, and the 3′-end of RNA (but not DNA) can be phosphorothioated by RNA ligase and dpCp(S) (Eckstein, 1985).
The non-bridging phosphorothioates can be incorporated into the backbone of nucleic acids by chemical or enzymatic methods (Zon and Stec, 1991). Effective analytical and preparative chromatography (reverse phase HPLC) methods for purification and analysis of phosphorothioate oligonucleotides were developed for their clinical evaluation as antisense agents (Zon and Geiser, 1991; Zon and Stec, 1991; Padmapriya et al., 1994; Gerstner et al., 1995). Due to the present unavailability of a chemical stereo-specific synthesis, synthetic oligonucleotides with n phosphorothioate residues are mixtures of 2n possible diastereisomers (Rp and Sp). Only the stereoisomers of oligonucleotides with a few phosphorothioate residues can be separated and purified by HPLC (Chu and Orgel, 1990). Enzymatic synthesis of phosphorothioate polynucleotides (RNA or DNA) using appropriate templates, polymerases, and thiotriphosphate nucleotides yields stereo-specifically the Rp isomers. The purification protocols of phosphorothioate polynucleotides longer than 50-mers are usually based on metal-affinity, chromatography, and electrophoresis (see below).
Phosphorothioate physico-chemical properties. Replacing an oxygen by sulfur in a phosphate reduces the charge on the remaining oxygens while increasing the negative charge on sulfur. Protonation of phosphorothioates occurs preferentially on oxygen rather than sulfur since phosphorothioates are stronger acids (have lower proton affinities) than phosphates (Frey and Sammons, 1985; Liang and Allen, 1987).
The charge polarization (O=P—S−) and diastereoisomer linkages in phosphorothioates could have implications for structures and stability of their complexes, both antisense and triplex (Frey and Sammons, 1985). Furthermore, the larger sulfur atom and longer P—S bond (in comparison to P—O) could lead to steric crowding, especially in triplexes (Latimer et al., 1989; Hacia et al., 1994). Also, since sulfur forms weaker hydrogen bonds than oxygen, the phosphorothioate modification could disturb or modify specific H-bonding interactions with the first water shell in the major groove of duplexes. (Hacia et al., 1994). Biophysical studies demonstrate that oligonucleotides exclusively containing all −Rp or all −Sp, or random diastereoisomer mixtures of POS linkages, have different affinities for complementary single- and double-stranded sequences (Kim et al., 1992; Hacia et al., 1994; Lacoste et al., 1997). DNA duplexes formed by phosphorothioate (POS) oligonucleotide derivatives are usually less stable than those made of unmodified oligonucleotides (Latimer et al., 1989; Kibler-Herzog et al., 1991; Jaroszewski et al., 1992; Kanehara et al., 1995; Hashem et al., 1998), depending on the number of POS linkages, and their stereochemistry and location in the DNA sequence. For example, in the duplex, [d(GGsAATTCC)]2, the “inward” oriented (when S atom points into the major grove) Rp phosphorothioate isomer had a Tm only 1° C. below that of the all-phosphodiester duplex, while the “outward” oriented Sp isomers had almost no effect on Tm (Zon and Geiser, 1991). The lower binding affinity of short (<15 nt) all-POS antisense oligonucleotides can be offset simply by extending their complementary sequences by several nucleotides (Zon and Geiser, 1991).
In triplex-forming oligonucleotides (TFO), a small number of POS linkages at or near the ends (see
In fact, because they have high resistance towards exonucleases, oligodeoxynucleotides with POS-capped ends should be superior to normal oligonucleotides for experiments in vivo (Alumni-Fabbroni et al., 1994).
Relatively new bifunctional oligonucleotide probes, combining antisense and triplex-forming domains (see
Reactivity of the phosphorothioates versus purines in nucleic acids. Although both the N7- and N1-positions of adenosine are known to bind platinum, Strothkamp and Lippard (1976) showed that in the reaction between [(terpy)PtX]n+ and AMPS at rf≦1, binding occurred exclusively at sulfur. In contrast to the experiments with polynucleotides, reaction at the base was indeed observed when the platinum was present in excess over AMPS (Strothkamp and Lippard, 1976). Further evidence of high affinity of a sulfur towards platinum came from the base pre-binding experiments: in which [(terpy)Pt(NO3)]+ was allowed to react with base (presumably through N7/N1 atoms of adenosine) in the presence of 150-fold excess of both AMP and UMP for 45 min at 25° C. However, addition of 1 mole of UMPS per mole of platinum into this mixture resulted in the quantitative formation of [(terpy)Pt(UMPS)] within a few minutes suggesting that rearrangement from Pt-base binding into Pt—S binding occurred (Strothkamp and Lippard, 1976).
The selectivity of [(terpy)ptX]n+ towards compact phosphorothioate moieties over the bulky base is enhanced by the large terpyridine ligand coordinated to Pt2+. A sterically hindered system around platinum should result in a sharp decrease in reaction rate (Howe-Grant and Lippard, 1980) between [(terpy)Pt] and a base, especially in structured polynucleotides having base stacking interactions, involvement in H-bonding, or tertiary structure cages. Also, the strong intercalative binding of terpyridineplatinum(II) complexes to double helix decrease their reactivity towards bases participating in the stacking interaction (Lippard, 1980). The acidotriaminoplatinum structure of [(dien)PtCl]Cl (
Since sulfur reacts with the [Pt-amine] compounds mainly through the directly substitution of the Cl− ligand without prior aquotation, the reaction between [(dien)PtCl]+ and glutathione (HSDR) is nearly independent of the [Cl−] concentration whereas the reaction with GMP can be completely inhibited at high concentration of NaCl (Reedijk, 1991). In contrast, [(dien)Pt(H2O)]2+ will almost selectively react with GMP (Reedijk, 1991). Elmroth and Lippard (1994) showed that the rate of platination of GG site (through N7) in d(TTTTTTTTGGTTTTTTT) by cis-[Pt(NH3)(NH2C6H11)Cl(H2O)]+ is approximately 35-fold higher than that in the dinucleotide d(GG), and only about 3-fold less reactive than the phosphorothioate site in d(TTTTTTTSTTTTTTT), irrespective of whether the oligonucleotide was single- or double-stranded oligonucleotide structure.
In the reaction with platinum reagents, Gn clusters (where n32) are the most reactive sites in DNA (Bruhn et al., 1990; Lepre and Lippard, 1990; Gonnet et al., 1996). At GGG (and longer G tracts), the N7 of the central residue is the most nucleophilic site(s), and [(dien)PtCl]+ preferentially attacks this site (Yohannes et al., 1993).
Theoretically, if one [L3Pt]2+ group (where L is an amino ligand) is already attached to a guanine, it is expected to repel a second one reacting with an adjacent guanine. However in model experiments using [(NH3)3PtCl]+ (
Therefore, in contrast to homopyrimidine phosphorothioate oligonucleotides, where selective platination of POS residues eventually can be obtained, the platination of the oligonucleotides containing both phosphorothioate group(s) and Gn clusters will result in a mixture of P—S and Pt-N7(Gua) adducts.
Further details of the invention are illustrated in the following non-limiting examples.
Tumor necrosis factor alpha (TNFα) plays an important role in the immune response to infection. However, exaggerated production of this cytokine (also called cachetin) can lead to cytotoxicity, organ failure and death in the case of septic shock (see, e.g., Beutler and Grau, Crit. Care Med. 21:S423-435 (1993) and Dinarello, J. Infect. Dis. 163:1177-1184 (1991)). Moreover, TNFα, along with interleukin-1, has been shown to mediate the pathogenesis of chronic inflammatory joint diseases such as arthritis (Probert et al., Eur. J. Immunol. 25:1794-1797 (1995)) as well as cachexia, or wasting syndrome (Tracey and Cerami, Ann. N.Y. Acad. Sci. 569:211-218 (1989)). Antibodies directed against TNFα have been shown to protect against the lethal effects of septic shock and cachexia (Beutler and Grau (1993), supra and Tracey and Cerami (1989), supra), indicating that TNFα is a good candidate for antisense and antigene therapy.
As presented below, we have tested the effectiveness of the above described method for down-regulating TNFα expression in macrophages both in vitro and in vivo. Specifically, we have tested the ability of preformed antisense-TNFα mRNA complexes to block ribosome passage during translation in vitro and have compared our current constructs with the antisense sequence alone. We have also tested the effectiveness of our constructs for inhibiting TNFα production in macrophage-like cell lines and macrophages in mice using delivery procedures already proven for this system.
A. Construction and Structure of Antisense Molecules
Chimeric RNAs consisting of the minimal hairpin ribozyme sequence plus antisense and triplex-forming moieties (ATR 1) targeted to the intended target sequence of TNFα mRNA were designed as shown schematically in
Self-processing at 37° C. of primary pre-ATR 1 RNA during transcription at the sites flanking the mature ATR 1 sequence (see “cleavage/ligation” sites in
The linear species (R4) can fully pair with the target upon binding, as long as the folded structure of the ribozyme can open. After pairing, the ends are again able to approach each other, perhaps aided by formation of a triplex region, as shown in
B. Improved Binding Characteristics of ATR 1 Antisense Oligonucleotides
An RNA molecule containing the first 709 nt of the TNFα mRNA (designated herein as “TNF1”) was transcribed using the pGEM-4 vector system and T7 RNA polymerase and was employed as a target for various antisense RNA molecules. Specifically, an autoradiogram was made after electrophoresis on 6% denaturing (8 M urea, 2 mM EDTA) polyacrylamide gel of the gel-purified linear ATR 1 RNA (“R4” form) in equilibrium with its circular “R1” form (lanes 1-6 of
We found that ATR 1 antisense RNAs could form ultrastrong complexes with the target TNFα RNA molecule. These complexes appeared to be stable enough to be detected as several individual bands with close mobility when examined by denaturing electrophoresis in 6-8% denaturing polyacrylamide gels containing 8M urea, 2 mM EDTA at 45° C. (see
In control experiments we used the AT RNA which, as described above, lacks the hairpin ribozyme domain but retains the sequences capable of forming the specific complex(es) with the TNF1 RNA (see
When the RNA complexes were dissolved in 45% formamide and 5 mM EDTA, heated at 95° C. for 2 min. and subjected to polyacrylamide gel electrophoresis under denaturing conditions, only a small amount of the ultrastrong complexes between TNF1 and [32P]-labeled ATR 1 RNA was detected, and all less stable complexes inducing the smearing were abolished under these conditions (
Kinetic and gel-shift analysis of the formation of complexes between TNF1 RNA and the antisense RNAs showed that initial binding (presumably through ordinary Watson-Crick sense-antisense base pairing) is rapid, relatively weak, and occurs with roughly the same binding constants and rates of formation for both AT and ATR 1 RNAs (data not shown). These primary complexes can be detected by gel electrophoresis under nondenaturing (but not denaturing) conditions.
C. Assessment of the Ability of ATR Antisense RNAs To Block Ribosome Scanning or Translation
Since we have demonstrated greatly enhanced stability of binding between ATR 1 antisense RNA and a TNFα RNA target, we next tested how well this strong complex is able to block ribosome scanning of the 5′ untranslated region of an mRNA template or translation from that template. We, therefore, constructed a TNFα-luciferase fusion (designated herein as “PTS” for “Promoter-Target-Start Codon”) by inserting a TNFα target sequence flanked by a T7 promoter transcription enhancer and an AUG translation start codon into the NcoI site of pGL3 Control Vector (Promega, Madison, Wis.), a luciferase vector containing an SV40 translation promoter and enhancer (see
The PTS fusion was pre-incubated in 50 mM Tris acetate (pH 7.5)/10 mM magnesium acetate, either alone or in the presence of a 10-to-40-fold molar excess of either ATR 1 or AT antisense RNA for 1 hr at 37° C. The resulting sense-antisense complexes were then added to a rabbit reticulocyte lysate (Promega, Madison, Wis.). Following in vitro translation of the complexes, luciferase production was measured. A T7 promoter-luciferase expression plasmid (designated herein as “PS”) lacking the TNFα target sequence was used as a further control.
The results of these experiments are presented in
D. Other Anti-TNF ATRs
The ATR 16a, ATR 16b and ALR 229 constructs (see
The ability of the new ATR 16a, ATR 16b and ALR 229 constructs to form strong complexes with TNFα RNA is shown in
E. Kinetics of Strong Complex Formation
To understand relative rates of hybridization and strong complex formation, we removed aliquots of ATR-target complexes after different times of incubation together and electrophoresed one-half of each aliquot on a nondenaturing gel (to detect hybridized complexes, either strong or weak) and the other half on a denaturing gel. For ATR 16a, ATR 16b and ALR 229, the inital kinetics were almost identical for both complementary hybridization and strong complex formation (FIGS. 12A-C), showing that hybridization is the rate-limiting step. In contrast, the analysis of binding ATR 1 with appropriate TNF target (
F. Delivery of ATR 1 Antisense Constructs to Macrophages for Down-Regulating TNFα
Effective delivery of biologically active RNA (Malone et al., Proc. Natl. Acad. Sci. USA 86:6077-6081 (1989)) and ribozymes (Sioud et al., J. Mol. Biol. 223:831-0835 (1992)) has been demonstrated for cells of hematopoietic origin. Lipofectamine (DMRIE/DOPE; Life Technologies, Bethesda, Md.) has been used to introduce our constructs into murine peritoneal macrophages both in vivo and in vitro because previous studies had shown it to be superior to a variety of other lipid formulations, with low toxicity (Kisich and Erickson, J. Leukocyte Biol. Suppl. 2:70 (abstract) (1991a) and Kisich and Erickson, FASEB J. 4:1860 (abstract) (1991b)).
For example, 10 μg of 32P-labeled hairpin ribozyme (HPR) was complexed with either Lipofectin (DOTMA/DOPE), DMRIE/DOPE, or Lipofectamine and administered i.p. to mice in a volume of 1 ml. Macrophages were harvested after 8 hours and lysed in 97% formamide 5 mM EDTA, 0.1% SDS. The lysates were analyzed by gel electrophoresis and phosphor-imaging to calculate the number of intact HPR molecules per macrophage. The results are presented in
As shown in
To produce responsive macrophages, 1 ml of aged sterile fluid thioglycollate broth (Difco, Detroit, Mich.) was injected i.p. into 6-week-old female C57b1/6NCR mice 3 days before peritoneal lavage. The resulting peritoneal exudate cells (PEC) will be obtained by lavage using Hanks balanced salt solution (HBSS), plated at 1×106/well in 24-well plates with Eagle's minimal essential medium (EMEM) with 10% heat-inactivated fetal bovine serum. After adhering for 2 hr, the wells were washed to remove nonadherent cells.
For in vitro transfection of macrophages or RAW264.7 cells (a macrophage-like cell line), Lipofectamine was diluted 1:4 with HBSS in polystyrene tubes, vortexed, and ribozyme construct were added at a 2.2-3.3:1 [DOSPA:RNA] phosphate charge ratio (Lipofectamine consists of DOSPA and DOPE in the w/w ratio of 3:1). After vortexing again, an amount of the mixture containing 1 μg of RNA were then added immediately to serum-free EMEM-rinsed cell cultures and allowed to incubate for 3 hr.
For in vivo transfection, the transfection reagents were prepared as described above except that the amounts were scaled up so that 10 μg RNA is used per mouse. After vortexing, 1 ml of the resulting [liposome:RNA] complexes were loaded into 1-cc syringes fitted with 30-gauge needles and then injected i.p. into mice that has previously been treated with thioglycollate to recruit responsive macrophages. Macrophages were harvested 3 hr later by peritoneal lavage with HBSS. The exudates were plated at 1×106/well in 24-well plates and allowed to adhere for 2 hr, then washed with HBSS to remove nonadherent cells.
After transfection, macrophages isolated as described above, or RAW264.7 cells were stimulated with 100 ng/ml lipopolysaccharide (LPS) to induce TNFα production. Supernatants were sampled at 0, 2, 4, 8, and 24 hr post-LPS stimulation and stored at −70° C. until quantitation, which were done by a TNFα-specific ELISA (Biosource International).
G. Development of Vectors For Endogenous Delivery of ATR 1 Antisense RNAs
To demonstrate the feasibility of using ATR antisense RNAs in a gene therapy setting, we have incorporated the genes encoding these molecules into eukaryotic expression vectors. The strategy for construction of the mammalian high expression DNA vectors for ATR generation is as follows. Single copy or concatomer copies of the ATR DNA templates are generated by PCR reactions and cloned into the CMV promoter driven pS65T-GFP-C1 vector (Clontech Labs, Palo Alto, Calif.) downstream from the GFP (green fluorescent protein) expressing region. The T7 promoted vector used for our ATR in vitro synthesis was used as DNA template for generation of mammalian cloning sequences. Using a 5′ overhang primer, restriction cloning sites for EcoRI and BglII and a eukaryotic stop codon are introduced upstream of the ATR sequence, eliminating the T7 promoter. Single ATR copy expressing vectors are constructed by cloning of the ATR template into the EcoRI-BamHI site in the MCS of the GFP vector. Concatomer copy expressing vectors are generated by creating a head-to-tail multimeric ligation between compatible cohesive ends of BglII head and BamHI tail sites. Head-to-head and tail to tail ligations are inhibited by having BglII and BamHI enzymes present in the ligation mixture (at 100 mM NaCl to avoid “star” activity). Head-to-tail ligations will not be cleaved by these enzymes. The final ladder product is isolated from acrylamide gel electrophoresis and cloned into the BglII-BamHI site of the GFP vector. In-frame directional clones are selected by the characteristic of being cleaved by these two enzymes.
The expression constructs will be introduced into the murine macrophage-like cell line, RAW264.7, via electroporation (at 960 μFd and 230 V, which has been previously shown to be optimal for this cell line) or lipofection with Lipofectin complexes with the DNA at a 2:1 charge ratio. Expression levels of the ATR antisense RNAs will be assessed by Northern analysis or RT-PCR (Sambrook et al. (1989), supra). Efficacy of the plasmid expressed triple helix antisense ribozymes will be assessed following stimulation of the transfected cells with lipopolysaccharide, followed by ELISA for secreted TNFα in the supernatants. Constructs that prove effective in this transient expression assay may then be incorporated into adenoviral or adeno-associated viral vectors (Kozarsky and Wilson, Curr. Op. Gen. Dev. 3:499-503 (1993) and Xiao et al., Adv. Drug Del. Rev. 12:201-205 (1993)) for assessment of in vivo efficacy. Further optimization of the basic ATR cassette (ATR 1 without the antisense and triplex-forming sequences), if required, will be carried out by deletion analysis and isolation of improved variants from partially randomized sequence libraries. Finally, antigene applications, in which double-stranded DNA is targeted, will also be tested.
H. Conclusion
Our data revealing the superior stability of the padlock RNA-target complex suggest that padlock antisense RNAs can wind around the target RNA strand and topologically link themselves to the target RNA molecules through catenation, in a manner similar to a real padlock. The “lock” is evidently the hairpin ribozyme domain, since AT RNA cannot form such a strong complex with the TNF1 target. Circularized probe molecules should be restricted in one-dimensional diffusion along the target RNA in nondenaturing conditions because of the TNF1 target's secondary and tertiary structure. However, Nilsson et al. (1994), supra, showed that circular oligonucleotide probes are free to travel considerable distances along the target strand during denaturation. In agreement with these findings, we found that the ATR 1 antisense RNAs complexed with the 396-nt longer (in comparison with the 3′ end of TNF1) target “TNF2” RNA were more resistant to denaturing conditions than complexes with the shorter TNF1 RNA target (data not shown) (the sense sequence was 563 nt from the 5′ ends of both target RNAs).
We found that covalent circularization (self-ligation) of the hairpin ribozyme domain in ATR 1 antisense RNA is not strictly required for formation of the strong complex with the TNF1 target. Thus, as discussed above, we showed that linear ATR 1 RNA molecules having 5′-[32P]-phosphate and 3′-OH ends (i.e, the “R4” form) failed to form the covalent circle R1 RNA. However, this “termini misphosphorylated” RNA molecule also could form strong complexes with the TNF1 target despite the fact that covalent cleavage should obviously result in the strongest padlock. More recent results suggest that a fraction of ligation-competent ribozyme-target complexes are significantly more stable, suggesting that these are covalently linked.
Since the structures of the hairpin ribozyme required for both self-cleavage and self-ligation were shown to be essentially identical (Chowrira et al. (1993), supra and Butcher and Burke (1994), supra) the covalent bond itself at the cleavage/ligation site presumably does not play a role in stabilization of the catalytically active ribozyme structure. A similar feature has been shown for circularly permuted tRNAs, which can form the same structure even when their nucleotide chains are interrupted by placing nicks in the middle of helices or in anticodon loops (Pan et al., Science 254:1361-1364 (1991)). Coaxial stacking across the cleavage sites is probably a factor in stabilizing these structures (Walter et al., Proc. Natl. Acad. Sci. USA 91:9218-9222 (1994)). Recent results (Butcher and Burke (1994), supra) indicate that the hairpin ribozyme adopts a stable, magnesium-dependent tertiary structure where the sequences adjacent to the cleavage/ligation site are likely involved in non-Watson-Crick base pairing interactions.
The ability of various anti-TNFα antisense constructs to inhibit the secretion of TNFα from RAW264.7 cells was determined. Specifically, 2×105 RAW264.7 cells were treated with 4.5 μg of the antisense construct ATR 1, ATR 16a, ATR 16b or ALR 229 or control RNA (m101) as described in Section I-F above. The RNA was complexed with Lipofectamine at a 3:3:1 charge ratio for 2 hours in 1 ml DMEM. TNFα levels in supernatents were measured by specific ELISA at increasing intervals after stimulation with 100 ng/ml LPS. The results of these experiments are presented in
As shown in
The above experiments were repeated identically except that the lipid:RNA charge ratio of the transfection complex was lowered from 3.3:1 to 2.2:1. This resulted in improved results. This improvement is evident in the dose response curves shown in
These data demonstrate the following points:
1. A Lipofectamine:RNA charge ratio of 2.2:1 is much less toxic than the 3.3:1 ratio previously used, for example in
2. Target selection is not limited to homopurine blocks but can be any sequence. For example, ALR 229, which contains no triplex-forming sequence and is targeted at a non-homopurine sequence, is the most potent antisense of these molecules, with an IC50 of about 46 nM.
3. It is possible to efficiently target coding regions in cells using antisense molecules (ATR 1, ALR 229) that do not depend on cleavage of their target mRNA for their effectiveness (in contrast to hammerhead ribozymes, or antisense DNAs that rely on cleavage by RNase H). This has never before been achieved, to the best of our knowledge.
4. Our newer padlock RNA constructs are even better than our original ATR 1; hence the ATR approach can be further improved by design innovations. At the same time, the fact that three or four out of four constructs designed were effective in cell assays indicates that our basic design principles are quite general, rather than being limited to a few instances of accessible target sequences.
We chose our most effective padlock RNA in the cell culture assay to test for in vivo efficacy in mice (
In response to injury or infection, leukocytes adhere to endothelial cells lining the walls of blood vessels in the area and proceed to emigrate through the wall and into the affected tissue. This process is mediated by the cytokine-induced expression of several adhesion molecules on the endothelial cell surface, including members of the selectin family (P-selectin, E-selectin) (Lawrence et al., Cell 65:859 (1991)) and members of the immunoglobulin family (ICAM-1, ICAM-2 and VCAM-1) (Oppenheimer-Marks et al., J. Immunol. 147:2913 (1991)). VCAM-1 is induced by IL-1, IL-4 and TNF, and reaches maximal levels 10-14 h after cytokine treatment, remaining elevated for up to 72 h (Rice and Bevilacqua, Science 246:1303 (1989) and Masinovsky et al., J. Immunol. 145:2886 (1990)).
The VCAM-1 gene, which is present in a single copy in the human genome, contains 9 exons spanning approximately 25 kilobases of DNA. Exons 2-8 contain C2 or H-type immunoglobulin domains. At least two different VCAM-1 precursors can be generated from the human gene as a result of alternative mRNA splicing events, which include or exclude exon 5 (Cybulsky et al., Proc. Natl. Acad. Sci. USA 88:7859 (1991)).
Sustained elevation of levels of VCAM-1 is associated with several pathologies, including atherosclerosis, retinosis, inflammatory bowel disease and asthma (Smith et al., Am. Rev. Respir. Dis. 148:S75-78 (1993), Gosset et al., Ann. N.Y. Acad. Sci. 725:163-172 (1994) and Ohkawara et al., Am. J. Respir. Cell. Mol. Biol. 12:4-12 (1995)). VCAM-1 is induced in rabbit aortic endothelium in vivo within 1 week after initiation of an atherogenic diet and is expressed in rabbit atherosclerotic lesions in vivo (Li et al., Am. J. Pathol. 143:1551-1559 (1993)). In hypercholesterolemic rabbits VCAM-1 may participate in initial monocyte recruitment to prelesional areas of arterial endothelium (Libby and Clinton, Nouv. Rev. Fr. Hematol. 34(supp):S47-53 (1992)). Therefore, the gene is an excellent candidate for antisense therapy as described herein.
The first step in applying this novel approach to the VCAM-1 gene was to synthesize DNA templates for an ALR antisense RNA (VALR1) targeted to a 20-nt site on VCAM-1 RNA, overlapping the AUG initiation codon (nt 636-655 of the genomic sequence [Cybulsky et al. (1991), supra]). This site was identical to the target site of a phosphorothioate oligodeoxyribonucleotide that was found to have significant activity in suppressing expression of VCAM-1 in HUVEC cells (Bennett et al., J. Immunol. 152:3530-3540 (1994)). This other oligonucleotide was “ISIS 3792”, directed against the AUG codon. When ISIS 3792 was synthesized as a 2′-O-methyl derivative, which does not support RNase H cleavage when hybridized to a target, antisense activity was lost, indicating that ISIS 3792 depends upon RNase H cleavage for its activity in cells (Bennett et al. (1994), supra). From these results we inferred that the site on VCAM mRNA complementary to ISIS 3792 was accessible to hybridization in vivo, and that it would be a good test of the ability of padlock RNA constructs to stall ribosome progression without depending on RNase H cleavage for their effectiveness. The antisense sequences of VALR1 and ISIS 3792 as well as the complementary sequence of VCAM mRNA (with start codon underlined) are as follows:
To the antisense sequence ribozyme and linker sequences were appended so that the expected structure of the resulting construct upon binding to its target would be as shown in
Synthesis of the complete VALR1 ATR RNA, designated VALR1, was accomplished by T7 transcription of appropriate DNA templates as described above for the TNFα system. For a target site, we used VALRT 1, a 642-nt partial transcript of VCAM mRNA fused with a piece of pSP-luc+NF vector sequence. This RNA contained a 20-nt sequence around the AUG codon complementary to the antisense domain of the VALR 1 molecule (see figures), located 247 nucleotides (nt) downstream of the target 5′-end and 375 nt upstream of the 3′-end. VALR 1 was even more active than ATR 1 (TATR 1) in self-processing (self-cleavage) of its precursor RNAs but, in contrast to ATR 1, self-processed linear
VALR 1 RNA showed very little ability to circularize in the absence of a target. However, self-processing of the pre-VALR 1 RNAs in the presence VALRT 1 target resulted in formation of circular VALR 1 species, as detected by denaturing gel-electrophoresis. We also showed that VALR1 RNAs can form a strong complex with VALRT 1 RNA, stable after gel-electrophoresis in denaturing conditions (8 M urea, 2 mM EDTA, 45° C. for 2 hr.).
For testing the ability of VALR1 to inhibit VCAM-1 expression, control RNAs are synthesized that lack the catalytic hairpin ribozyme moiety or contain an inactive, mutated version of it, as well as ALRs that lack the target binding site. These controls allow an assessment of specificity of binding and the importance of strong binding and linkage for biological efficacy. Phosphodiester and phosphorothioate versions of ISIS 3792 are also used to permit head-to-head comparison of ALR constructs with the type of antisense molecules currently under development as pharmaceuticals.
VALR1 and other antisense constructs that show strong binding in cell-free assays, together with control RNAs, will be assayed for their ability to down-regulate VCAM-1 expression in human vascular endothelial cells (HUVEC) cells. If appropriate to understand HUVEC cell results, we will also perform in vitro translation of luciferase mRNAs that have been modified to carry the appropriate ALR target sequence in either the 5′ UTR or the coding region as described in Example I above. Inhibition of luciferase production in rabbit reticulocyte lysates from ALR target/luciferase fusion mRNAs which have been precomplexed with ALR RNA will indicate sufficient complex stability to inhibit translation. For testing blockage of translation inside the cell, we will transfect the above constructs as preformed plasmid-ALR complexes into HUVEC cells and monitor luciferase activity as with the cell-free procedure, comparing with activity using control RNAs or no antisense. In this case the fusion gene will be expressed from the SV40 promoter. In vivo testing will also be performed as described above for TNFα antisense molecules.
Because cancer cells are in many respects like their normal counterparts, virtually all potent anticancer drugs lack the requisite specificity toward cancer cells alone and are, therefore, highly toxic. As such, we herein describe a novel method for achieving cancer cell specificity by providing an therapeutic antisense agent with two independent levels of recognition. For the antisense agent to be active against a cell, recognition at both levels must occur. One level consists of Watson-Crick pairing between a target mRNA and the antisense sequence within the agent. The agent is designed so that stable duplex formation requires binding of an additional molecule that is abundant only in cancer cells. This cell-specific entity is the second level of recognition. The antisense target gene can be any gene whose overexpression is associated with tumor progression or metastasis, and whose down-regulation is expected to normalize growth control. Although many such proteins will find use in the present method, we will herein use the HER-2 gene as the antisense target. Repression of HER-2 in mouse tumors leads to suppression of tumor growth and longer survival of the mice; hence it is an attractive target for antisense therapy. The structure and sequence of the binary agent will be optimized through biochemical assays for tightness and specificity of binding, and if necessary through selection from randomized sequences. Then its effectiveness at blocking translation will be tested, first in an in vitro translation system and then in cultured tumor cell lines.
Clinical trials of antisense therapy for cancer are underway for at least two target genes relevant to cancer. However, a major limitation of this approach, and indeed of all drug therapy in current use for cancer, is the lack of ability to target the drug specifically to cancer cells. Thus, the goal of the presently described work is to employ a novel method for providing antisense therapy of breast cancer with this needed element of cell specificity. Treatment of breast cancer patients based on this approach will provide increased effectiveness and greatly decreased side effects. As described above, we have designed a ribozyme-based antisense agent intended to covalently link itself around its target mRNA after hybridizing to it. This linkage provided much greater strength of binding and potency in blocking translation. Here, however, we extend that approach to make the linkage dependent on the presence of c-myc, a nuclear protein present in elevated levels in breast cancer cells which binds to a specific DNA sequence. Thus, the antisense oligonucleotide binds to the mRNA target, wrapping around it due to the helical winding of the sense-antisense duplex. By bringing the ends closer together and reducing the conformational entropy of the molecule, this binding to the target permits the ends to pair with each other in an additional short, weak duplex which contains a binding site for c-myc (with its cofactor max) (see e.g.,
The aggressively growing, invasive cancer cells which can become life-threatening in late-stage cancers are the end products of a series of genetic alterations that usually occur over many years. In the case of breast cancer, the genetic alterations that have been identified are mostly amplifications of a small number of oncogenes, among which are c-myc and HER-2 (c-erb-B2) (Van de Vijfer and Nusse, Biochim. Biophys. Acta 1072:33-50 (1991) and Kozbor and Croce, Cancer Res. 44:438-441 (1984)). Amplification of either of these genes is associated with aggressive breast cancer and poor prognosis (Wong et al., Am. J. Med. 92:539-548 (1992)). Transgenic mice containing the c-myc gene driven by strong promoters develop adenocarcinomas of the breast during pregnancy (Stewart et al., Cell 38:627-637 (1984) and Leder et al., Cell 45:485-495 (1986)). Similarly, overexpression of HER-2 has been shown to enhance malignancy and metastasis phenotypes (Hung et al., Gene 159:65-71 (1995)). Repression of HER-2 by delivery of certain viral genes (adenovirus-5 E1a and SV40 large T antigen) into tumor cells in mice leads to suppression of tumor growth and longer survival of the mice (Hung et al. (1995), supra). Thus HER-2 is a very attractive candidate for antisense therapy, and c-myc is a useful marker for the most dangerous cells. Cancer differs from infectious disease in that the “infectious agents” are in most respects like normal cells; hence the greatest challenge in cancer treatment is finding cytotoxic or cytostatic agents sufficiently specific for cancer cells that they have minimal toxicity at therapeutic levels. We present here a novel approach to achieving the needed specificity by having two levels of recognition, analogous to a binary weapon. Thus, instead of attacking all cells that, say, are actively dividing, or that possess a particular cell-surface marker, the proposed agents down-regulate an appropriate target gene only upon binding to some molecule that is present mainly or exclusively in the cancer cell. The great power of the method is that the triggering molecule and the target gene can be (but are not required to be) completely unrelated. The target gene can potentially be any gene, even a housekeeping gene, although the highest level of specificity is achieved by choosing a target gene that is active mainly or exclusively in cancer cells. Here, we choose as the triggering molecule the phosphoprotein product of the c-myc gene, and HER-2 as the target gene. The mechanism of triggering is based on the padlock idea but where topological linkage requires stabilization by binding of a separate agent, the “clasp.” We propose to use c-myc as the clasp.
A novel feature of this two-tiered approach is that its effectiveness should be proportional to the product of the concentrations of two elements that are more abundant in breast cancer cells: c-myc protein and HER-2 mRNA (or DNA, in the antigene version of the approach). Since the HER-2 gene is often amplified by as much as 10-fold, and the abundance of c-myc may be similarly elevated in some cancer cells, the therapeutic index of our approach is expected to be much larger than current therapies.
This approach lends itself well as an adjunct to other, standard therapies. Because c-myc overexpression is associated with poor prognosis of breast cancer, it might be expected that the resistance of tumor cells to standard chemotherapeutic agents may correlate with c-myc expression. This was found to be the case with cis-platin: Treatment of cis-platin-resistant cells in culture with c-myc antisense oligonucleotides reversed the resistance, possibly by increasing uptake of cis-platin; and even in nonresistant cells that express c-myc, there was a synergistic cytotoxic effect (Mizutani et al., Cancer 74:2546-2554 (1994)). Synergistic effects have also been seen for combination c-myc-P53 antigene therapy (Janicek et al., Gynecol. Oncol. 59:87-92 (1995)).
Like other members of the myc family, c-myc functions in cell proliferation and differentiation (Vastrik et al: Crit. Rev. Oncog. 5:59-68 (1994) and Penn et al., Semin. Cancer Biol. 1:69 (1990)). It has been shown to be both necessary and sufficient for normal resting cells to enter the cell cycle. In some circumstances, it also mediates apoptosis (Cherney et al., Proc. Natl. Acad. Sci USA 91:12967-12971 (1994), Saito and Ogawa, Oncogene 11:1013-1018 (1995) and Shi et al., Circulation 88:1190-1195 (1993)). A phosphoprotein, c-myc is localized to the nucleus and is involved in the transcriptional regulation of several genes, including ornithine decarboxylase, p53, prothymosin α and ECA39. It forms a heterodimer with a related protein, max, through their homologous helix-loop-helix and leucine zipper domains; and the dimer binds to DNA much more sequence-specifically than does either protein alone (Blackwood and Eisenman, Science 251:1211-1217 (1991)). The heterodimer transactivates target genes through binding to the sequence CACGTG. Transactivation is relatively insensitive to orientation or position of this sequence relative to the gene being activated (Packham et al., Cell Mol. Biol. Res. 40:699-706 (1994)).
A number of studies have documented effects of treating cells with antisense oligonucleotides directed against c-myc. For example, treatment of T-cell hybridomas interferes with activation-induced apoptosis (Shi et al. (1993), supra). Treatment of colonic carcinoma cells inhibits their colony-forming capacity (Collins et al., J. Clin. Invest. 89:1523-1527 (1992)). Treatment of estrogen-dependent (MCF-7) breast cancer cells with 10 μM c-myc antisense oligonucleotide resulted in 95% inhibition of c-myc protein expression, a 75% reduction in estrogen-stimulated cell growth, and a cytostatic effect also on estrogen-independent MDA-MB-231 cells (Watson et al., Cancer Res. 51:3996-4000 (1991)). Similar reduction in c-myc levels and growth inhibition was achieved at lower oligonucleotide concentrations (<1 μM) by conjugation with poly(L-lysine) (Degols et al., Nucleic Acids Res. 19:945-948 (1991)). Antigene, triplex-forming oligonucleotides complexed with polyamines have been shown to suppress c-myc expression in breast cancer cells (Thomas et al., Nucleic Acids Res. 23:3594-3599 (1991)).
An oligonucleotide designed to form a triplex with a G-rich sequence in the promoter region of HER-2 has been shown to inhibit transcription factor binding (Noonberg et al., Nucleic Acids Res. 22:2830-2836 (1994)) and transcription in vitro (Ebbinghaus et al., J. Clin. Invest. 92:2433-2539 (1993)).
Full-length c-myc binds sequence-specifically to its target site only as a heterodimer with the related protein max (Blackwood and Eisenman (1991), supra). To avoid having to use both c-myc and max in our cell-free assays, we will take advantage of the fact that a truncated version of c-myc consisting of the basic, helix-loop-helix, and leucine zipper domains binds to the same site as the c-myc/max dimer.
Candidate antisense constructs will be tested for c-myc-dependent binding and translation inhibition first in a cell-free system and then tested for biological effects on cultured breast cancer cells. The goal of these prototype experiments will be a general procedure for down-regulating any gene in the presence of any given protein.
To accomplish the above, the following will be performed. First, the sequences HER-5′, HERMYC1, and HERMYC2 as shown in
Next, the binding characteristics and kinetics by polyacrylamide gel-shift assay using 5′-32P-labeled padlock oligonucleotides will be verified. The DNAs will be annealed by heating and slowly cooling to 2° C., then electrophoresed at different temperatures ranging from 4° C. to 37° C. in the presence of MgCl2 to mimic intracellular concentrations of available Mg2+.
Next, the stability of the complexes will be verified by measuring the melting temperatures of padlock oligonucleotide HERMYC1 with target HER-5′ and HERMYC1 with HER-5′ by UV absorbance, ramping the temperature from 2° to 45° and back down at 0.5°/min in 25 mM Tris HCl (pH 7.4), 100 mM KCl, 1 mM EDTA. We will repeat in the presence of truncated c-myc protein (2, 10, and 100-fold stoichiometric excess). If the Tm is not above 37° in the presence of c-myc and below 20° in its absence, we will carry out an in vitro selection on the partially randomized HERMYC1R and HERMYC2R as shown in
Next, a T7 promoter fused to HER-5′ will be inserted into the NcoI site of pGL3 Control Vector (Promega, Madison, Wis.), a luciferase vector containing an SV40 promoter and enhancer. We will perform in vitro translation and 5′ capping using T7 RNA polymerase and capping reagent (Ribomax kit, Promega). We will then test the ability of HERMYC1 and HERMYC2 to block in vitro translation of the resulting RNA (as monitored by luciferase-dependent light production) using a rabbit reticulocyte lysate system, in the presence and absence of truncated c-myc. We expect truncated c-myc-dependent blockage in both cases.
Because several parameters may need optimizing for the padlock/target complex to have the best balance of low stability in the absence of the clasp and high stability in its presence, it may prove advantageous to select the best sequences from a randomized library of RNA sequences. Therefore, the selection may follow the scheme shown in
In vitro translation assays for ribozyme obstruction will be performed as described in the above example, using a fusion construct between HER-2 mRNA and luciferase. Controls will include oligos having scrambled and sense sequences in place of the antisense sequence, and clasp sequences that cannot bind c-myc. Before addition of an rabbit reticulocyte lysate translation mixture, oligos will be incubated in 50 mM Tris acetate (pH 7.5)/10 mM magnesium acetate, either alone or in the presence of a 10- to 40-fold molar excess of in vitro-transcribed (T7) HER-2-luciferase fusion constructs. Luciferase will be assayed according to the instructions from the translation kit manufacturer (Promega). A T7 promoter-luciferase expression plasmid lacking the target sequence will be used as a further control.
For cell testing, we will use the phosphorothioate versions of the successful oligonucleotides from the previous experiments. We will then expose estrogen-dependent MCF-7 breast cancer cells or other appropriate c-myc-overexpressing cells to this oligonucleotide or control oligonucleotides with and without estrogen, and assay for HER-2 mRNA, protein production, and cell proliferation. If appropriate, we will compare with estrogen-independent MDA-MB-231 cells, which constitutively express high levels of c-myc. We will use a cationic lipid vehicle: lipofectamine or lipofectin (Life Technologies), or dioleoylphosphatidylethanolamine (DOPE) together with a cationic cholesterol derivative (DC cholesterol) previously shown to be less toxic for down-regulation of HER-2 expression through a gene therapy approach (Hung et al. (1995), supra). We will also try the unmodified DNA oligonucleotides, since in some cases they survive intact long enough to be effective for treating cells in vitro.
Our clasp-dependent translation blockage can also be applied to blocking transcription; i.e., as an antigene agent. We will test the antigene capabilities of our locking system by targeting the HER-2 promoter region between the CAAT box and the TATA box, at the following sequence:
which has been shown to inhibit transcription factor binding (Noonberg et al., Nucl. Acids. Res. 22:2830-2836 (1994)) and transcription in vitro (Ebbinghaus et al., J. Clin. Invest. 92:2433-2539 (1993)). Delivery and assays will be the same as for the antisense oligos. Attractive alternative target genes include H-ras and VEGF. A proposed padlock for the latter is shown in
With the rapid increase in available DNA sequence information, nucleic acid-based diagnostics is a subject of intense interest. Improvements in the efficiency of sample preparation, nucleic acid amplification, and detection would permit greatly increased use of such methods for routine diagnostic purposes. This example describes ways in which target binding can activate RNA catalysis, leading to improved methods of detection or amplification of target molecules. The most widely used method for amplifying DNA prior to detection is the polymerase chain reaction (PCR). Alternative isothermal methods involve a sequence of enzymatic steps, with the attendant complexity, costs of the protein enzymes, and risk of contamination during multiple tube openings. Here we propose innovations in isothermal amplification based on the use of RNA as an amplifiable probe and the ability of RNA to catalyze its own cleavage and rejoining reactions. By taking advantage of RNA catalysis, we can eliminate requirements for all protein factors except an RNA polymerase and reduce the number of tube, openings. Additional innovations in hybrid capture and wash procedures further increase the speed and ease of automation. The procedure lends itself to closed-tube, multiplex fluorescent detection during amplification, permitting rapid screening of many different targets while minimizing risks of exposure to pathogens or laboratory contamination by amplified targets.
The ultimate goal is to design a scheme for nucleic acid-based diagnostics that can be used to detect either DNA or RNA, is sensitive, rapid, requires no thermal cycling, and readily lends itself to automation. Our proposed scheme is based on the use of RNA catalysis and Qβ-replicase for amplification. This RNA-dependent RNA polymerase will catalyze exponential replication of an RNA molecule possessing appropriate end sequences, without the need for primers or thermal cycling. Following the work of Tyagi et al., Proc. Natl. Acad. Sci. USA 93:5395-5400 (1996), we separate the end sequences by dividing the substrate RNA into two halves, called replication probes; hence amplification can proceed only if the two replication probes are ligated together (
In light of the above, we will design and construct, using randomization and selection procedures, a latent hairpin ribozyme derivative becoming catalytically active only after binding to a target sequence (
Nucleic Acid-Based Diagnostics. The detection and quantitation of DNA and RNA are increasingly important techniques. They are used for diagnosing infectious diseases caused by viruses and microorganisms, detecting and characterizing genetic abnormalities, and identifying genetic changes associated with cancer or various types of treatment. Further uses are in detecting pathogenic organisms in the medical supply (such as blood banks), food and environment samples. As research tools these techniques are used in genetics, virology and microbiology, as well as in forensic sciences, anthropology and archeology. With their increasing importance comes a need for their simplification and improvement in order to be used routinely in many varied applications.
A common method for detecting genomic material is to quantitate specific nucleic acid sequences through hybridization with nucleic acid probes. These probes can carry radioactive or other types of labels, including ligands, which can interact with detecting moieties, e.g. streptavidin or digoxigenin. However, the sensitivity of nucleic acid hybridization is limited by the specific activity of the probe. Even under optimal conditions, direct hybridization methods can detect only down to 106 nucleic acid molecules (Horn and Urdea, Nucl. Acids Res. 17:6959-6967 (1989), which is not sufficient for many desired applications. Moreover, the most sensitive hybridization assays usually lack features required for routine applications—safety, economy, convenience and speed.
One solution of this limited sensitivity is exponential amplification of the target sequence. This can be carried out by either temperature-cycle assays such as the polymerase chain reaction (PCR) and ligation chain reaction, or isothermal procedures such as transcription-mediated amplifications and the restriction nuclease/DNA polymerase method. Alternatively, hybridization can alter another component of the reaction so as to make it amplifiable; examples include linear amplification methods such as induction of an enzyme reaction to produce a fluorescent product, or exponential amplification of reporter RNA through the use of Qβ-replicase (Chu et al., Nucl. Acids Res. 14:5591-5603 (1986)).
In practice, PCR, although providing very high sensitivity, has several limitations: (1) the occurrence of false positives generated by hybridization of primers to homologous sites in non-target DNA, (2) the presence of PCR inhibitors in specimens, and (3) its inability to directly amplify RNA due to its thermal lability. These drawbacks, together with market considerations of license fees for the use of patented PCR technology and the significant cost of thermal cyclers, have stimulated a search for alternatives. Prominent among the newer techniques is the isothermal, exponential amplification of recombinant RNA probes by Qβ-replicase. Although the PCR and the Qβ-replicase assays have similar sensitivity (Lizardi et al., Biotechnology 6:1197-1202 (1988)), the Qβ-replicase assay is simpler, faster and less expensive than PCR. The substrate for Qβ-replicase is RNA rather than DNA, and RNA combines the dual functions of hybridization probe and amplifiable reporter. In this proposal we make use of a unique capability of RNA, autocatalysis, to eliminate the need for two protein enzymes used in a promising current amplification scheme (Tyagi et al. (1996), supra) employing Qβ-replicase.
Qβ-replicase is an RNA-dependent RNA polymerase from the coliphage QB. It is capable of replicating the single-stranded Qβ RNA genome in infected cells while ignoring the huge excess of bacterial RNA, and similar specificity has been observed in in vitro assays (Haruna and Spiegelman, Science 150:884-886 (1965)). As little as one molecule of template RNA can in principle initiate its exponential amplification by Qβ-replicase without any need for primers. The first step is the template-directed synthesis of the complementary strand, after which the two complementary strands spontaneously separate, permitting each to be the template for another round of complementary strand synthesis. As long as there is a stoichiometric excess of the enzyme, the number of replicated RNA molecules increases exponentially. After the number of RNA replicas equals the number of enzyme molecules, the RNA amplification continues linearly. The final amount (more than a billion copies) of the synthesized RNA (typically, 200 ng in 50 μl in 15 min at 37° C.) is so large that it can be easily detected by simple colorimetric techniques (Lizardi et al. (1988), supra.
Other than the natural Qβ genome, MDV-1 RNA, only a few RNAs have been found to exhibit template activity for Qβ-replicase (Munishkin et al., J. Mol. Biol. 221:463-472 (1991)). Although Qβ-replicase has been studied for more than 30 years, the precise RNA structure requirements for replication remain obscure. The only obvious common element is extensive secondary structure within the single strands, which probably serves to prevent or inhibit duplex formation after replication (see
Two developments led to the possibility of using Qβ-replicase, despite its highly specialized template requirements, for amplification of arbitrary RNA sequences. First is the discovery that an oligoribonucleotide fragment (up to 58 nt in length) can be inserted within the sequence of 221-nt MDV-1 (+) RNA (a naturally occurring template for Qβ-replicase) between the nucleotides at positions 63 and 66 without interfering with its replicability (Lizardi et al. (1988), supra). This capability permits the incorporation of functions in addition to being an amplifiable reporter. The second development is the design of latent RNA probes which cannot be amplified unless they hybridize to their target (EP-A-707 076 and Tyagi et al. (1996), supra), so-called “smart” probes. These permit very low background in hybridization assays, since signal generation is strictly dependent on the presence of target RNA.
One smart probe approach is to divide the amplifiable reporter RNA into two separate molecules neither of which can be amplified by itself, because neither contains all the elements of sequence and structure that are required for replication. The division site is located in the middle of the embedded probe sequence. When these “binary probes” are hybridized to adjacent positions on their target, they can be joined to each other by incubation with an appropriate ligase, generating an amplifiable reporter RNA. Nonhybridized RNA probes on the other hand, because they are not aligned on a target, have a very low probability of being ligated.
In another approach (EP-A-707 076), latent Qβ-replicable template has been created by extending on the 5′ end of MDV-1 RNA resulting in inhibition of its replication by Qβ-replicase. A ternary hybrid formed between this latent substrate, a second RNA probe, and a target RNA produces an autocatalytic RNA structure (hammerhead ribozyme) which cleaves the 5′ extension from the latent template in the presence of divalent cations, thereby converting it to an efficiently replicating form and effecting its release from the support. This approach is interesting as a first attempt to use the catalytic potential of RNA molecules in nucleic acid-based diagnostics. However, it has two disadvantages: It is limited to target sequences that contain the element GAAA (required to generate an active hammerhead ribozyme) and unintended spontaneous RNA cleavage (and, therefore, activation of replication) can readily occur through transesterification, catalyzed by the same divalent metal ions that are required as catalytic cofactors for the ribozyme.
In this approach, we combine the best features of these two approaches by employing a hairpin ribozyme construct for the target-dependent-ligation of two halves of a split substrate for Qβ-replication. Our approach imposes no sequence restrictions on the target RNA, and because spontaneous ligation can be made much rarer than spontaneous RNA cleavage, the background of false positives should be very low. Moreover, because the hairpin ribozyme can catalyze both ligation and cleavage (see below), it can play dual roles of target-dependent ligase and specific endonuclease. We use the endonuclease activity for release from a solid support after hybrid capture, replacing a ribonuclease H(RNase H) used in the Tyagi et al. procedure. (As mentioned above, capture permits a purification step prior to amplification, which reduces background.) We further suppress background by requiring that each step of the procedure be dependent on recognition of the target; this is accomplished through hybridization with five separate RNAs. Actually, this approach works for both RNA and ssDNA targets.
The HPR has distinct domains for substrate binding and catalysis that interact through specific tertiary contacts (see above). These domains can be on the same RNA molecule or on separate molecules (Feldstein et al., Gene 82:53-61 (1989)). The release of cleavage products permits binding to another substrate for further cleavage or ligation. Both HPR and Qβ-replicase require magnesium ions for activity in dilute solution.
Ability of HPR to cleave and ligate adjacent, distant, and unattached substrates. We have demonstrated the ability of two HPR constructs, HPR1 and HPR2, to cleave and ligate adjacent or distant substrate sequences on the same RNA strand (reaction in cis), as well as to ligate substrates on separate molecules (reaction in trans) through formation of specific RNA-RNA complexes. A 150-nt DNA template encoding the T7 promoter, a spacer sequence, and the pre-processed sequence of the minimonomer hairpin ribozyme was transcribed to generate the pre-HPR1 RNA (161-nt). This template was transcribed by T7 RNA polymerase in the presence of the non-radioactive nucleoside triphosphates and/or [α]32P] CTP. Denaturing polyacrylamide gel electrophoresis of transcription products revealed the presence of several RNA species (
Catalysis by freezing and ethanol. Extensive characterization of individual forms generated by self-processing of HPR T7 transcripts revealed that either freezing alone or exposure to >40% ethanol alone results in ligation of linear forms in the absence of divalent cations (Kazakov et al., submitted)). Actual freezing was required, since ligation did not occur in supercooled but unfrozen solutions at −21° C.
Both freezing and ethanol treatment were able to produce yields of ligated product (up to 85%) that exceed the yield (˜50%) in normal aqueous solution. Since the key step in our scheme for amplification by Qβ replicase is ligation of the replication probes (see below), we have the option of incorporating a freezing step if needed for maximal sensitivity.
We describe here procedures for analysis of sample of cells to be analyzed for pathogens through detection of mRNA; with an initial denaturation step, a similar protocol would work for DNA targets, suitable, for example, for detection of genetic variants. The general scheme is outlined in
A sample of cells to be analyzed for pathogens is dissolved by incubation in 5 M guanidine thiocyanate for 60 min at 37° C. This treatment lyses cells, inactivates enzymes, frees DNA and RNA from intracellular structures, and weakens RNA secondary structures (Pelligrino et al., Biotechniques 5:452460 (1987)). Lysates are adjusted to reduced guanidine thiocyanate concentration (Buffer A: 2M guanidine thiocyanate, 400 mM Tris-HCl (pH 7.5), 0.5% sodium N-lauroylsarcosine, 0.5% BSA); this solution continues to block nuclease activity and promotes RNA-RNA hybrid formation without interference from cell debris (Tyagi et al. (1996), supra). Capture probe RNA1 is added and allowed to hybridize for 60 min. The target RNAs complementary to the capture probes are then captured by adding 20 μl of a suspension of streptavidin-coated paramagnetic particles (Promega) and incubating at 37° for 10 min (
The noncomplementary nucleic acid molecules, excess capture probes, and cellular debris are removed by thorough rinsing, first with Buffer A (4 times), then with Buffer B (5 mM MgCl2, 66 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40 (Sigma)) to remove guanidine thiocyanate (4 times). For each wash cycle, the mixture is vortexed, and beads are gathered to the side of the tube with a magnet, and the solution is removed and replaced with a fresh wash.
After the last wash step, a final aliquot of Buffer B is added along with RNAs 2 and 3 (
To remove from further reaction any excess capture probes and uncleaved complexes, the beads are drawn aside with a magnet and the solution is transferred to another tube containing RNAs 4 and 5, buffer C (15 mM MgCl2, 45 mM Tris-HCl (pH 8), 100 μM ATP, 600 μM CTP, 600 μM UTP, and 600 μM GTP) and Qβ-replicase (6 μg, Vysis).
From this point forward, no further manipulations are required; the remaining steps proceed in sequence automatically. RNAs 4 and 5 hybridize to a pair of sites located 500 nt from the binding site of the HPR domain E, and the 2′-3′-cyclic phosphate end of RNA4 and the 5′-OH end of RNA5 make up a substrate pair that can bind and be ligated by the HPR. They can form a complex through looping of the target RNA as shown in
Synthesis of RNAs with appropriate ends. The replication probes must have 5′-OH and 2′,3′-cyclic phosphate ends in order to be ligated by HPR domain E. The simplest way to achieve this is the automatic scheme shown in
The above described scheme will be tested using as target a sequence from the pol gene of HIV-1. Domain E will bind to nt 4668-4682 of the HIV genome (Tyagi et al. (1996), supra), the capture probe will bind to nt 4716-4760, the left replication probe to nt 4577-4588, and the right to nt 4607-4618. We will first test for the efficiency of the individual steps by using 32P-labeled target or probes and following the recovery at each step.
We must also design a derivative of HPR that has catalytic activity dependent on target binding. In the normal HPR, the catalytic domain (E) is stabilized by a short Watson-Crick helix capped by a loop. The loop can be replaced by three additional base pairs and full activity is maintained. However, other alterations of the stability of this helix can strongly affect both thermostability and catalytic activity of hairpin ribozyme. To make catalytic activity dependent on target binding, we substitute a Y-branch for the closed loop and select a sequence that will not support catalytic activity unless the target is accurately paired.
The scheme for selecting sequence NNNN is an in vitro selection and amplification procedure (Breaker and Joyce, TIBTECH 12:268-275 (1994)) based on sequential RNA-catalyzed cleavage and ligation reactions (Berzal-Herranz et al., Genes Dev. 6:129-134 (1992)). Two RNA molecules are synthesized that, in the presence of an oligonucleotide target fold into the hairpin ribozyme-like structure shown in the right panel of
An alternative method is to synthesize a series of molecules having from 0 to 6 A-U base pairs and 0 to 4 G-C pairs in place of NNNN, testing them individually for target-dependent catalytic activity.
Having obtained good candidates for the target-dependent catalytic moiety, the next step will be to demonstrate its ability to cleave a separate RNA (in trans) if the two RNAs are hybridized to adjacent sequences on a target RNA. For this purpose, we will construct DNA templates for the transcription of target RNA spanning the region of HIV to which all the probes bind (nt 4500-4800). Using the biotinylated probe shown in
The next step is to test the ability of the target-tethered ribozyme to ligate the Qβ replication probes (constructed with appropriate ends as described above) and achieve amplification in the presence of the replicase. If necessary, we will test the ability of Qβ replicase to use our probes as substrates by synthesizing a short complementary RNA to hold the ends together and performing ligation with DNA ligase.
Finally, we will combine the above steps and test the ability of the ribozyme to switch from cleavage of the capture probe to ligation of the replicase probes in the presence of the target. If needed, we will adjust lengths and/or AT/GC composition of helical stems surrounding cleavage/ligation sites if needed.
To test for sensitivity and background, prepare simulated diagnostic samples by using a dilution series of T7-transcribed target RNA so that samples will contain as little as 1 molecule of target. α-[32P]-CTP will be included in the Step 3 (see
For detection of HIV-1 pol gene RNA in mammalian cells, we will use a COS-like Monkey kidney cell line (CMT3) stably transfected with plasmids pCMVgagpol-rre-r (containing gag and pol genes) as a model (Anazodo et al., J. Clin. Microbiol. 33:58-63 (1995)). These cells contain a biologically active provirus that expresses HIV-1 pol mRNA. Simulated clinical samples will be prepared as a dilution series of cells expressing HIV-1 pol RNA in a constant population of nonexpressing cells. The number of expressing cells per 100,000 nonexpressing cells will range from zero and 1 to 10,000. Finally, actual clinical specimens will be tested. We also will test the effectiveness of the sample preparation procedure on blood and urine samples to see how well guanidine thiocyanate alone will protect against the effects of nucleases in those fluids. Additional nuclease inhibitors and/or the use of 2′-amino or other appropriate modifications to RNA probes will be employed if necessary.
As an alternative detection procedure, we will simply add a small amount of ethidium bromide or propidium iodide and follow the increase in fluorescence in a fluorometer as the Qβ reaction proceeds. Fluorescence detection will be the method of choice for commercial version of this method. The fluorophore can be either using a single intercalating dye as here, or a panel of oligonucleotides each conjugated to a different fluorophore distinguishable by their emission or excitation maxima. Each oligonucleotide would be complementary to a different replication probe and a different target, permitting multiplex amplification and detection of many different targets in the same sample. With appropriate reaction containers, the fluorescence could be measured without opening the container, reducing risk of contamination of the lab by amplified product and permitting destruction of the sample immediately after measurement.
Plunno et al., Anal. Chem. 67:2635-2643 (1995) have described a fiber-optic DNA sensor for fluorometric determination of nucleic acids, involving tethering of oligonucleotide probes to the surface of quartz fiber-optic filaments. Target nucleic acids hybridized to the probes cause enhanced fluorescence of intercalated ethidium bromide, which is detected through epifluorescence based on excition and emission through total internal reflection along the fiber. We plan to test the use of capillaries bearing tethered probes instead of streptavidin beads for hybrid capture. Unlike solid fibers, capillaries permit reaction and washing steps to be done without pipetting, much as DNA is synthesized on a solid-phase column. Detection could still be performed through fiberoptic means, since the capillary would provide for total internal reflection of exciting and emitted light at least as efficiently as with solid fibers.
Enhancement of ligation by freezing. The efficiency of ligation can be increased from about 50% to about 85% by freezing the solution to −5° for 15 min. We will try this procedure to see if the added efficiency warrants the additional step.
Relaxing stringency. The procedures described above provide the maximum level protection from false positive signals, by requiring independent target recognition by five separate RNA molecules in order for amplification to proceed. Such high “stringency” may not be necessary, especially for certain applications. Thus, it may not be necessary to have the replication probes bind to the target, as long as the catalytic activity of Domain is strictly dependent on target binding. The replication probes could be tethered to Domain E either covalently or via hybridization or metal coordination to provide efficient target-dependent ligation in this case.
Sensitivity. The sensitivity of this assay depends on the efficiency of all the steps leading to amplification of the target RNA. In the version of Tyagi et al. (1995), supra, the proportion of target molecules that resulted in amplifiable product was 2.5%. The main source of loss was the ligation step using T4 DNA ligase, which operates inefficiently on RNA and produced only 8% ligated product. In contrast, typical HPR derivatives such as HPR1 exhibit ligation efficiencies of 50% and as high as 85% upon freezing. Even with an overall efficiency of 2.5%, Tyagi et al. (1995), supra, could detect the presence of 100 but not 10 molecules of target. We anticipate that the higher yield of HPR ligation will increase the sensitivity to less than 10 molecules.
The other important characteristic of an effective diagnostic technique is the background of false positive events. False positives in this procedure come from ligation events that occur in the absence of hybridization to the target. Qβ-replicase can occasionally continue polymerization across a gap between replication probes that somehow are juxtaposed to each other, resulting in an amplifiable reporter RNA. Tyagi et al., (1995), supra, found that the only significant source of such events was the beads, which apparently sometimes bound the probes in such a juxtaposition. By including the capture step, concentration of free replication probes was so low that not even a single amplifiable RNA was generated from a sample containing 100 uninfected cells, although a single infected cell in 100,000 was detected. Our procedure retains this feature, and has the added security that to generate an amplifiable RNA molecule, four separate RNAs must be brought together by hybridization to a single target molecule. Domain C is catalytically inactive without some stabilizing interaction, such as with a binding protein (Sargueil et al., Biochemistry 21:7739-7748 (1995)) or our target. While there is some probability that a ligatable complex could occur between a target-bound domain C and RNAs 4 and 5 that were not bound to the target, the creation of such a complex would be a third-order event and the individual binding energies between partners are low. In any case, such unlikely events would not lead to false positives, because they still depend on domain C binding to the target.
All of our proposed nucleic acids are RNAs and hence are sensitive to cleavage by contaminating ribonucleases. The inclusion of 2 M guanidine thiocyanate in the cell lysate mixture was sufficient to prevent cleavage of target RNA to a degree that would significantly reduce the sensitivity of the assay. Since the lengths of RNA required in our procedure are comparable, this precaution is likely to be sufficient here also. If further reduction in nuclease cleavage is desirable, several options are available. Additional inhibitors of RNases, such as SDS, phosphate ions, and RNase inhibitors such as RNasin (Promega) could be included in the capture step. Also, RNAs 1-3 (and perhaps 4 and 5) can be synthesized with 2′ modifications such as amino groups that render them RNase resistant. These modified nucleotides can be present in all but a few positions and still permit efficient catalysis. Moreover, they can be synthesized by T7 RNA polymerase using appropriate nucleoside triphosphates.
We also may employ longer pairing stems in the substrate sequences for RNAs 4 and 5 and shorter ones for RNA 1, then adjust the temperature of incubation so that release of captured target is efficient while providing good yield of ligated replication probe. If necessary, we could employ a second Domain E, binding close to the binding sites of the replication probes, and also dependent on target binding for ligation activity.
The above discussion illustrates one manner in which binding of a target can activate a latent ribozyme to produce a signal for detecting the target molecule. Another embodiment of this idea is shown in
A more general embodiment of this idea is shown in
All stock solutions were prepared in water deionized by a Milli-Q apparatus (Millipore), and then filtered through 0.22 Am filter units (Nalgene). The contents of common stock buffer and solutions used were as follows:
Synthetic 16-mer oligodeoxyribonucleotide (TT) and its phosphorothioate derivatives (TST and STT) (1 micromole scale, GF grade) were obtained from Midland Certified Reagent.
Dried stocks of oligodeoxynucleotides were dissolved in 1×TE buffer to get a concentration about 10 μg/μl and then passed through the ‘Ultra free MC filter units’. 20 μl of these solutions were mixed with 20 μl of 2×FLS, loaded on the 20% denaturing polyacrylamide gel (1.6 mm), and then electrophoresed at 800 volts. The main oligonucleotide bands were located by UV shadowing of the gel, than cut out and extracted from the crushed gel slices by soaking into elution buffer (EU) at 37° C. for 2 h. The extract was passed through the microcentrifuge 0.22 μm filter, and the clear solution obtained was mixed with 4 vol. of absolute ethanol and kept overnight at −80° C. Precipitation of the purified oligonucleotides was completed by centrifugation 14,000 rpm at 4° C. for 10 min. Pellets were washed twice by 1 ml of absolute ethanol, dried, resuspended in 100 μl of 1×TE and passed through the microcentrifuge 0.22 μm filter. When not being in use, the oligonucleotide solutions were kept at −20° C. Aliquots (25 μl) of these solution were diluted to 1 ml and UV spectra were measured to determine their concentrations. The 5′-end labeling of the oligodeoxynucleotides using T4 polynucleotide kinase (Richardson, 1981) were done using labeling protocol described below:
32P-labeled oligonucleotide species were purified (and analyzed) by electrophoresis through 20% denaturing polyacrylamide gels. Immediately before loading onto the gels, the solutions were mixed with equal volumes of 2×FLS, and heated for 2 min at 95° C. Individual oligonucleotide bands were located by autoradiography and isolated from the gels as described above.
Diethylenetriamine catalyzes platination of oligonucleotides. Various reaction mixtures (total volume 10 μl) were combined as following:
The reaction conditions, including reagent concentrations, temperatures and times of incubation, are indicated in the figure legends. The platination reactions were stopped by the addition of 1 μl of 1 M NaCl (Brabec et al., 1994), mixed with an equal volume of 2×FLS and analyzed by electrophoresis on 20% denaturing polyacrylamide gel.
In agreement with literature data (Wherland et al., 1973; Kasianenko et al., 1995) regarding low reactivity of anionic [PtCl4]2− towards nucleic acids, we found that K2[PtCl4] at 30 μM concentration does not affect the electrophoretic mobility of the 10 μM oligonucleotide TT or its derivative TST having single phosphorothioate (POS) linkage (
Plausible structure of platinum groups tethered to oligonucleotides. There are two possible pathways by which a bimolecular reaction proceeds first in such preformed [chloroplatinate-dien-oligonucleotide] complexes with high local concentrations of the reactants. The first pathway is a reaction between [PtCl4]2− and dienH22+ yielding [(dien)PtCl]+, although this reaction proceeds very slow even in highly concentrated solutions (Watt an Cude, 1968; Mahal and Van Eldick, 1987). The second possible pathway is direct reaction between [PtCl4]2− (or products of its aquotation) and nucleophilic atoms available in the oligonucleotide, followed by binding between the tethered platinum group and dienH22+ associated with the negatively charged nucleic acid surface. It is known that the products of the initial binding of [PtCl4]2− to polynucleotides are heterogeneous, unstable and very reactive (Chu and Orgel, 1990a; Kasianenko et al., 1995). Moreover, both POS sulfur and guanine N7 can additionally activate the tethered platinum groups due to their strong trans-influence (Howe-Grant and Lippard, 1980). To distinguish these two pathways, we studied an effect of increasing concentrations of Cl− on the platination of TT and TST oligonucleotides (
Interestingly, the presence of 1 mM NaCl in the reaction mixture does not reduce the platination yield (
What metal binding center is more reactive: POS or Gn clusters? The longer reaction time at 45° C. provides a higher yield of platination of both TT and TST oligonucleotides after 2 h incubation (
However, we obtained an evidence that the POS residues are more reactive than the GGGG cluster. We showed that the platination of all-phosphorothioate STT under the same conditions as for TT and TST oligonucleotides {30 μM K2[PtCl4], 10 μM oligonucleotide (molar ratio Pt:oligonucleotide=3:1) and 3 mM dien}forms a three-band ladder (
The additional positive charge of the platinum groups also allows an easy separation of platinated oligonucleotides from reaction mixtures by preparative electrophoresis. As an alternative isolation method we might consider ion-exchange column chromatography. We also suggest using of metallo-affinity chromatography on mercurated columns as a one-step method of purification of platinated phosphorothioate oligonucleotides.
Optimum conditions for platination of phosphorothioate and G-rich oligonucleotides:
The contents of the optimal platination mixtures (10 μl) is:
(* fresh solution in H2O pre-incubated for 1 h at 45° C. before adding to the reaction mixture).
In looking for the optimal conditions, we found prominent effects of a variety of components of reaction mixtures on the platination yield and number of products formed.
Dien. The higher the dien concentration, the better the yield of the platination. Compare, for example, the product yields in the presence of 1 mM dien and 3 mM dien (
Pt and oligonucleotide concentrations. For preparative bimolecular (second-order) reactions the concentrations of both components are important. In our case the amount of radioactive platinum (and therefore its concentration) is a limiting factor. We can not resolve this limitation by using an excess of oligonucleotide components (making of pseudo-first order reaction) because of possible oligonucleotide precipitation and problems with isolation of platinated oligonucleotides. We determined the allowable concentration range for K2PtCl4 to be 3 to 100 μM in 10 μl reaction mixtures (0.03 to 1 nmoles platinum); below that level we did not detect any platination of oligonucleotides for reasonable reaction time, whereas above that level we observed fast non-specific modification and precipitation of oligonucleotides. The optimal [Pt:oligonucleotide] molar ratio in reaction mixtures was determined to be useful in the range Of 1.5 to 6 (
K2PtCl4 pre-treatment. It was shown that the transition of [PtCl4]2− to its aquo-complexes [PtCl3(H2O)]− and [PtCl2(H2O)2]0 affected its binding with DNA (Kasianenko et al., 1995). Aquotation starts immediately in freshly prepared K2PtCl4 solutions, and we found that this process can affect on the reproducibility of our experiments. Therefore, we recommend pre-incubating the fresh stock solutions of K2PtCl4 (10 mM) for 1 h at 45° C. to complete this process before using for the oligonucleotide modification.
The foregoing description details specific methods which can be employed to practice the present invention. Having detailed such specific methods, those skilled in the art will well enough know how to devise alternative reliable methods at arriving at the same information in using the fruits of the present invention. Thus, however, detailed the foregoing may appear in text, it should not be construed as limiting the overall scope thereof; rather, the ambit of the present invention is to be determined only by the lawful construction of the appended claims. All documents cited herein are expressly incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60056742 | Aug 1997 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09137059 | Aug 1998 | US |
| Child | 11228705 | Sep 2005 | US |
