Patent Application
20070105108
RNA-based technologies have become increasingly prominent in research and biotechnology since the discovery of naturally existing antisense RNAs, catalytic RNA (ribozymes), techniques for selection of aptamers from random libraries of RNA (SELEX), and RNA interference (RNAi) (Sullenger & Gilboa, 2002). A major problem for any RNA agent that relies upon efficient hybridization to complementary sequences is to identify which target sites are accessible in vivo. Consequently, the rational design of effective RNA agents can be slow and inefficient.
Over the past 25 years, modified antisense oligonucleotides (Lichtenstein & Nellen, 1998; Stein & Krieg, 1998; Crooke, 2001) and artificial ribonucleases, including ribozymes and deoxyribozymes (Rossi, 1999; Opalinska & Gewirtz, 2002; Scherer & Rossi, 2003) have been used with variable success for antisense-mediated gene silencing (ASGS) through the targeting of messenger or viral RNAs. Recently, a practical approach to the exploitation of RNAi, the use of small interfering RNAs (siRNA), has emerged (Tuschl, 2002; Zamore, 2002; Paddison & Hannon, 2003; Shi, 2003). Optimized antisense compounds and cleaving ribozymes (or DNAzymes) can work as effectively as siRNA although higher concentrations of the ASGS agents are required (Al-Anouti & Ananvoranich, 2002; Braasch & Corey, 2002; Brantl, 2002a; Opalinska & Gewirtz, 2002; Grunweller et al., 2003; Miyagishi et al., 2003; Vickers et al., 2003). A number of naturally occurring antisense RNAs that have been identified in both prokaryotes (Brantl, 2002b, Brantl & Wagner, 2002; Wagner et al., 2002) and eukaryotes (Vanhée-Brossollet & Vaquero, 1998; Kumar & Carmichael, 1998; Yelin et al., 2003), and have been shown to be highly specific and efficient in ASGS. Antisense modulation of gene expression in human cells has been suggested to be a common regulatory mechanism (Carmichael, 2003; Yelin et al., 2003). Moreover, recent results concerning the complexes formed between antisense RNA and target RNA provide direct evidence for mechanistic links between ASGS and RNAi, both of which involve double-stranded RNA (Di Serio et al., 2001; Martinez et al., 2002; Tijsterman et al., 2002; Holen et al., 2003). Artificially-designed antisense RNAs have also been proven to be powerful tools to downregulate the expression of targeted genes (including genes that are poor targets for small interference RNAs) in both prokaryotes and eukaryotes (Lafarge-Frayssinet et al., 1997; Upegui-Gonzalez et al., 1998; Varga et al., 1999; Chadwick & Lever, 2000; Terryn & Rouze, 2000; Ji et al. 2001; Wang et al., 2001; De Backer et al., 2001, 2002, 2003; Ji et al., 2002; Martinez et al., 2002; Yin & Ji, 2002; McCaffrey et al., 2003).
SiRNAs inhibit translation through cleavage of their targets, but the mechanism of action of antisense agents is not completely understood. There is evidence that translation inhibition by antisense agents is not always the result of lowering the levels of target mRNA (Probst and Skutella, 1996). In fact, many efficient antisense oligonucleotides function either via steric blocking of the translation machinery (including at regulatory protein binding sites) or by inducing a conformational change in the target RNA, rather than by RNase H-mediated cleavage of the target (Stein, 2000; Toulme, 2001; Braasch and Corey, 2002). These noncleaving modes of action enable the use of antisense techniques in applications for which the elimination or cleavage of mRNA targets would be undesirable, such as regulation (or redirection) of alternative splicing (Volloch et al., 1991a, b; Taylor et al., 1999; Zeng et al., 1999; Gong et al., 2000; Sierakowska et al., 2000; Mercatante et al., 2001) or intracellular imagining of gene expression (Paillasson et al., 1997; Pan et al., 1998; Politz et al., 1998; Tavitian et al., 1998; Chartrand et al., 2000; Molenaar et al., 2001; Pederson, 2001; Dirks et al., 2003). Thus, the ASGS methods provide viable complementary approaches to RNAi technologies (Lavery and King, 2003; Scherer and Rossi, 2003).
The need for antisense agents that are more potent, selective, robust and reliable is widely recognized, which has led to the development of new agents with higher binding affinity and selectivity, as well as schemes for their selection using sequence libraries (Sohail & Southern, 2000). It appears that the affinity of antisense sequences for their target sites (Walton et al., 1999) and the kinetics of hybridization (Patzel & Sczakiel, 2000) are the most important factors in the efficacy of antisense agents. Recently, several new nucleic acid derivatives, including N3′-P5′ and morpholino phosphorodiamidate, 2′-O-methoxyethyl and 2′-fluoroarabino-nucleic acid, have been shown bind strongly to target RNA without resulting in cleavage of the target (Toulme, 2001; Braasch & Corey, 2002; Heasman, 2002; Kurreck, 2003). However, all of these nucleic acid molecules are artificial and, therefore, cannot be expressed by transcription, which is a very good way of providing the high intracellular concentrations that are required for efficient translation inhibition. DNA and RNA may have less stability in vivo than their chemically modified derivatives, but they both can be efficiently and systemically expressed in situ from appropriate PCR templates, plasmids and viral vectors. RNA has certain advantages over DNA—it can be more efficiently expressed in cells than DNA (e.g., using the U6 or H1 pol III promoters) (Noonberg et al., 1994), and RNA-RNA duplexes are more stable than DNA-RNA hybrids (Beckmann & Daniel, 1974; Roberts & Crothers, 1992; Lesnik & Freier, 1995; Landgraf et al., 1996; Wu et al., 2002). When antisense RNAs anneal to complementary sequences of the target transcript, they may affect RNA stability or translation directly, or cause the target transcript to be retained in the nucleus, or stimulate an RNA interference and/or PKR-interferon response (Kumar & Carmichael, 1998). RNA-RNA duplexes, although they are not substrates for RNase H, can be degraded (with some constraints on the antisense sequences) by Dicer, RNase L and RNase P ribonucleases (Kumar & Carmichael, 1998; Terryn & Rouze, 2000; Di Serio et al., 2001; Martinez et al., 2002; Tijsterman et al., 2002; Holen et al., 2003; Pulukkunat et al., 2003; Raj & Liu, 2003). Although at one time the concept of using RNA as a gene inhibitor seemed unlikely due to concerns about the RNA stability in cells, there is now clear evidence of the potential efficacy of RNA-based drugs (Sullenger & Gilboa, 2002).
The development of circularizable nucleic acids is a significant advance in the fields of nucleic acid-based therapeutics and hybridization probe diagnostics. Because of the helical nature of nucleic acid duplexes, circularized probes are wound around the target strand, topologically connecting the two polynucleotides (
The specificity and efficacy of padlock probes depends on the fidelity and efficiency of a DNA ligase enzyme for the ligation of substrate sequences on different templates. However, DNA ligase does not always discriminate against mismatched sequences. Both terminal and internal mismatches, which frequently occur upon hybridization (especially in GC-rich regions), can be joined by DNA ligase (Goffin et al., 1987; Wu & Wallace, 1989a, b; Luo et al., 1996; Pritchard & Southern, 1997; James et al., 1998). In vitro selection experiments of sequences that can be ligated most efficiently by T4 DNA ligase (using substrate sequences with randomized nucleotides) showed that many of the selected sequences had one or more mismatches even at the ligation junction (Harada & Orgel, 1993; James et al., 1998; Vlassov et al., 2004). However, it should be noted that the accuracy of DNA ligase could be enhanced under certain conditions (e.g., at elevated temperatures using thermostable DNA ligases, high sodium chloride and low ligase concentrations) (Luo et al., 1996). It is possible to optimize ligation conditions for an individual sequence to enable SNP detection in vitro (Luo et al., 1996), but these conditions will not generally be optimal for other sequences or oligonucleotide libraries. Also, ligation of DNA termini aligned on RNA targets occurs with very low efficiency (Nilsson et al., 2000; Nilsson et al., 2001), thus limiting their use as translation inhibitors.
Padlock probes are typically 70-100 nt in length (Antson et al., 2000) and, therefore, require laborious purification by preparative HPLC and gel-electrophoresis since the specificity and efficacy is absolutely dependent on the purity of the material. The proportion of imperfect synthetic oligonucleotide sequences increases with length as a result of incomplete nucleotide deprotection, depurination followed by strand scission during deprotection, premature termination of synthesis, and dephosphorylation (Kwiatkowski et al., 1996). Padlock probes missing terminal nucleotides or 5′-phosphate cannot be specifically ligated. These shorter or dephosphorylated sequences will also compete for target sequence, thus, reducing the yield of perfect ligations. Alternatively, padlock probes can be prepared by asymmetric PCR (Antson et al., 2000; Myer & Day, 2001). A drawback of this method is that DNA polymerases either tend to add (if they are exonuclease-minus) or to remove nucleotides at the 3′-end of the synthesized strand (if exonuclease-plus) (Antson et al., 2000). In both cases, padlock probe circularization is inhibited due to the ligation requirement for perfect ends. The maximum reported yield of ligatable padlock probe sequences after careful optimization of PCR protocols using exonuclease-minus DNA polymerase is only 60-70% (Antson et al., 2000).
“Lassos™” are an additional class of nucleic acid molecules that can circularize around and form topological links to target molecules (
A previously-described RNA Lasso ATRL (
RNA Lassos have been successful in inhibiting gene expression in several model systems (Johnston et al., 2000, 2002; Seyhan et al., 2001). Specifically, it has been shown that the enhanced stability of binding between Lassos and TNFα target can provide better inhibition of protein synthesis than ordinary antisense RNA. The 20-nt TNF target sequence was fused to a luciferase reporter gene and a T7 RNA polymerase promoter was attached upstream to create the cassette T7-TNF-Luc (
To assess Lassos' in vivo efficacy in cultured cells, the RNA Lassos, complexed with cationic lipids, were delivered to a macrophage-like cell line, RAW264.7, testing for their ability to inhibit TNFα secretion following stimulation of the cells with lipopolysaccharide (LPS). Different Lasso constructs (including ATR1 and four others) targeted to different sites on the TNFα mRNA (including both 5′-UTR and coding sequences) were tested. A Lasso construct (M101), lacking any sequence complementary to TNFα mRNA, was used as a negative control. The Lassos had an inhibitory effect that was evident for at least 24 hours after LPS stimulation, reducing TNF secretion up to 90% at a level (10 μg) that caused no nonspecific toxicity (IC50 of 46 nM). No inhibition of secretion was observed with M101 at similar levels. In other experiments in which multimers of ALR229 were delivered through a cytoplasmically replicating viral vector based on Semliki Forest virus (SFV), about 95% inhibition was seen (Johnston et al., 1998).
Not all Lassos have been found to be effective, presumably because of their differing abilities to access their target mRNA sites and to circularize around the target.
In general, the efficacy of antisense-based gene inhibitors is dependent on both position and sequence of the target sites, but this efficacy does not always correlate with RNA target site accessibility (Far & Sczakiel, 2003). The use of antisense agents is complicated by the lack of convenient, reliable methods for selecting the most sensitive target sequences. In most cases, potential target sites must be screened individually to find one that allows efficient knockdown of gene expression. But the ‘trial-and-error’ methods for identifying accessible sites are laborious and expensive. Consequently, attempts have been made to select superior sites through computer prediction and in vitro combinatorial approaches (Stull et al., 1996; Bruce & Lima, 1997; Lima et al., 1997; Matveeva et al., 1997; Milner et al., 1997; Ho et al., 1996, 1998; Patzel & Sczakiel, 2000; Lloyd et al., 2001; Sohail & Southern, 2000; Wrzesinski et al., 2000; Allawi et al., 2001; Pan et al., 2001; Scherr et al., 2001; Sczakiel & Far, 2002; Yang et al., 2003). However, there is frequently little correlation between sequences identified as accessible through these procedures and sequences that are truly active as target sites in living cells (Laptev et al, 1994; Yu et al., 1998; Sczakiel & Far, 2002). This incongruity may reflect the different folding of the RNAs within the microenvironments of the living cell versus in cell-free media or as a result of their interactions with RNA-binding proteins in cells. Because there is a poor track record of these methods in predicting accessible regions in vivo, some effort has been made to perform target site selection using randomized antisense sequence libraries (Lieber & Strauss, 1995; Kramer et al., 1997; Kruger et al., 2000; Kawasaki & Taira, 2002). Thus, there is a need for better methods of selecting target sites for nucleic acid-based targeting agents.
There is also a need for improved Lassos that exhibit greater effectiveness in forming a topological linkage with a target and control over sequence specificity in target binding.
The invention provides a novel class of allosterically-regulated polynucleotide molecules (also termed “Lassos”) that have advantages over currently-existing nucleotide binding agents which may be used, for example, for gene target imaging, detection, and inhibition. These molecules can undergo target-dependent self-circularization to become became topologically linked with nucleic acid targets. Previously-described Lassos contain a non-allosterically-regulated hairpin ribozyme (HPR) catalytic domain that can spontaneously adopt either a linear or circular conformation. Allosteric regulation in the present invention is achieved by converting a target-binding antisense sequence into a “sensor” sequence that binds to a regulatory nucleic acid sequence, serving to either block catalysis by a catalytic domain or lock the Lasso into an open conformation in the absence of target binding, thus preventing self-circularization of the Lasso prior to hybridization with the target. The sensor-antisense sequence is designed so that it has higher affinity to the complementary target sequence, which serves as a catalytic “effector,” than to the regulatory element. Upon binding of the sensor-antisense sequence to the to the target-effector sequence, a conformational (structural) rearrangement occurs, allowing circularization of the Lasso around the target, via formation of either a covalent bond (ligation) between two nucleotide residues of the Lasso or strong non-covalent bonds such as H-bonds, stacking interactions and coordination bonds involving metal ions (or a combination of two or more of these types of bonds and interactions).
This scheme of allosteric regulation is similar to the ‘TRAP’-like mechanism previously described for the cleavage reaction catalyzed by the hammerhead ribozyme (Porta & Lizardi, 1995; George et al., 1998; Burke et al., 2002). However, such a scheme has not been previously described to regulate cleavage and ligation activity of hairpin ribozyme. Moreover, the present invention is unique in using an allosterically regulated ribozyme for circularization of a polynucleotide around the target.
An ideal regulatory element must be sufficiently competitive to block both circularization of the Lasso and non-specific hybridization to the target, but not so competitive as to hinder formation of perfectly matched duplex between Lasso antisense and sense target sequences. In addition to the allosteric regulation, such competition may significantly decrease potential Lasso off-target binding. In other words, the regulatory sequence could function as a “stringency element,” increasing sequence specificity of target recognition and binding via “displacement hybridization” (Roberts & Crothers, 1991; Hertel et al., 1998; Bonnet et al., 1999; Ohmichi & Kool, 2000). A recent report of the high selectivity of oligonucleotide probes containing self-complementary elements to single-nucleotide mismatches or deletions suggest that even single-nucleotide mutation (SNP) discrimination is possible (Li et al., 2002).
Antisense sequences in Lasso constructs may be either rationally designed based on available experimental data or selected by an appropriately modified SELEX technique using a randomized Lasso library, as described in greater detail below. Such libraries may contain either fully random or the directed antisense libraries. A selected antisense sequence may be used to rationally design regulatory elements. In addition, other parts of a Lasso, including catalytic and non-catalytic sequences, or even both sensor and antisense sequences simultaneously, may be at least partially randomized to select/optimize the allosteric regulation and circularization activity. As discussed below, we have prepared allosterically-regulated Lassos against a model target, muTNFα mRNA, and demonstrated both their specific binding to the target and target-dependent circularization. Circularization provides very strong binding to a nucleic acid target, as well as increased resistance to exonucleases.
Circularized and topologically linked Lassos may be selectively amplified even at a trace amount by rolling circle amplification (RCA) or/and RT-PCR as described herein. Such amplification may be used for both detection of specific RNA targets and the selection of optimal Lasso constructs that bind these targets and circularize around target efficiently. Such strong and specific binding may be used for detection and/or inhibition of functions of a target molecule. As described in greater detail below, we have developed and demonstrated the feasibility of such a selection scheme.
To select for Lassos that can circularize efficiently around a target, randomized, Lasso libraries were prepared and exposed to an mRNA target. The resulting strong (stable) Lasso-target complexes were isolated, and the circularized Lasso molecules were selectively amplified by RT-PCR as shown in
Optimized Lassos can also be used as hybridization probes (e.g., for Northern blots, in situ hybridization, and microarrays), with utility in the fields of genomics, biodefense, forensics, microbiology, virology and oncology. Topologically linked Lasso-target complexes provide greatly increased binding strength while the sensor element responsible for allosteric regulation also provides higher sequence specificity compared to ordinary cRNA probes since it competes effectively for binding of the antisense sequence with mismatched targets, but is efficiently competed by matched targets. Rolling circle amplification (RCA) of the circularized probe by reverse transcription alone or reinforced by PCR provides very sensitive detection. Topologically linked probes can survive in a complex with circular or immobilized targets at high stringency. Because both washing and the RCA steps allow only probes that have undergone target-specific circularization to be detected, there is a significant enhancement in the signal to noise ratio. Alternative methods of detection that can be used include, for example radioactive, fluorescent, hapten, or enzymatic labels, or binding pairs such as biotin-avidin or streptavidin, which may be directly or indirectly incorporated into the probes during chemical or enzymatic synthesis or by post-synthetic modification. The selection approaches described above could rapidly provide probes which are capable of fast, specific hybridization to accessible target sites, target-dependent circularization, and topological linkage to a target.
In one aspect, the invention provides an allosterically-regulatable polynucleotide which is capable of specifically binding to a target nucleic acid molecule and circularizing around the target, forming a topological linkage.
In one embodiment, polynucleotides of the invention comprise a target binding sequence which is at least partially complementary to and capable of binding to a sequence of the target, and a catalytic domain which is capable of a catalytic activity that is inhibited or prevented from occurring in the absence of binding of the polynucleotide target binding sequence to the target. In the presence of binding of the target binding sequence to the target, the catalytic activity of the catalytic domain catalyzes circularization of the polynucleotide around the target, forming a topological linkage of the polynucleotide to the target. In some embodiments, the catalytic activity is a ligase activity and the catalytic domain catalyzes ligation between two nucleotide residues of the allosterically-regulatable polynucleotide. In one embodiment, the ligase activity catalyzes ligation between 5′ and 3′ ends of the polynucleotide. In another embodiment, the ligase activity catalyzes ligation between the 5′ end of the polynucleotide and a 2′ hydroxyl group of an internal nucleotide of the polynucleotide, thereby forming a “lariat” shaped structure around the polynucleotide. A lariat structure has a free, unligated end to which a detectable label may be optionally attached.
The catalytic domain of polynucleotides of the invention may include RNA or DNA residues or both, or analogs and/or modified forms of these nucleotides thereof. In some embodiments, the catalytic domain comprises, consists of, or consists essentially of RNA residues or analogs and/or modified forms thereof (e.g., the catalytic domain of a ribozyme, for example, comprising, consisting of, or consisting essentially of the catalytic domain of a hairpin ribozyme). In some embodiments, the catalytic domain comprises, consists of, or consists essentially of DNA residues or analogs and/or modified forms thereof (e.g., the catalytic domain of a deoxyribozyme).
In some embodiments, catalytic activity of the catalytic domain is inhibited by a regulatory sequence that is at least partially complementary to and binds to at least a portion of the target binding sequence, rendering the catalytic activity dependent on binding of the polynucleotide to the target. Circularization of the polynucleotide and topological linkage of the polynucleotide to the target are prevented in the absence of target binding, and are permitted upon binding of the polynucleotide to the target. In some embodiments, the conformation of the polynucleotide in the presence of the bound regulatory element prevents access of the catalytic domain to the substrate sequences required for circularization of the polynucleotide around the target.
In another embodiment, polynucleotides of the invention comprise a target binding sequence that is at least partially complementary and capable of specification binding to a target sequence, and circularization proceeds via noncovalent interaction between sequences of the polynucleotide, creating a loop or circular domain that encompasses the target binding domain. In this embodiment, a catalytic domain is not required. Allosteric regulation is accomplished by a conformational change in the polynucleotide upon target binding. For example, allosteric regulation may be achieved by alternative folding that partially occludes the target binding sequence in the absence of target binding. Upon binding of the polynucleotide to the target, the alternative interaction is disrupted, allowing base pairing to occur, circularizing the polynucleotide around the target and producing a loop to which the target is topologically linked (
The nucleic acid target may include RNA or DNA residues or both, or analogs and/or modified forms of these nucleotides thereof. In some embodiments, the target comprises, consists of, or consists essentially of RNA residues or analogs and/or modified forms thereof (e.g., mRNA). In some embodiments, the target comprises, consists of, or consists essentially of DNA residues or analogs and/or modified forms thereof (e.g., cDNA, genomic DNA). In one embodiment, the target is single stranded. In another embodiment, the target is double-stranded, and targeting may be via formation of a triplex or D-loop complex between the target and the polynucleotide of the invention.
Allosterically-regulatable polynucleotides of the invention may be prepared by chemical synthesis, or by in vitro or in vivo transcription from an expression vector. In one embodiment, the polynucleotides are transcribed in vitro using RNA polymerase, for example, phages T7, SP6, or T3. In another embodiment, polynucleotides are transcribed in the nucleus of a host cell, for example, by RNA polymerase II or III. The invention also provides allosterically-regulatable polynucleotides prepared by any of the methods described herein.
The synthetic or in vitro transcribed allosterically-regulatable polynucleotide can be either delivered to cellular targets either directly in liposomal complexes or they can be expressed in situ using plasmids or viral vectors. In the case of expression of allosterically-regulatable polynucleotide constructs in the nucleus by Pol II RNA polymerase (using an appropriate expression vector), the suppression of 3′ end processing of the polynculeotide could provide poly(A)-mediated, enhanced export of the polynucleotide to the cytoplasm. Pol 11-mediated polyadenylation may also provide additional nuclease resistance and help attract proteins with helicase activity that may help in binding to a structured mRNA target site (Kawasaki et al., 2002).
In another aspect, the invention provides a complex comprising an allosterically-regulatable polynucleotide as described above circularized around and topologically linked to a nucleic acid target molecule.
In another aspect, the invention provides methods for circularizing a polynucleotide around a target nucleic acid molecule, forming a topological linkage with the target. Such methods comprise contacting a target nucleic acid molecule with an allosterically-regulatable polynucleotide as described above, wherein binding of the polynucleotide to the target, via the target binding sequence of the polynucleotide, either alleviates inhibition of the catalytic activity of the polynucleotide or unblocks sequences required for circularization, thereby allowing topological linkage via circularization of the polynucleotide around the target to occur. The invention also provides methods for reducing the efficiency of transcription and/or translation from a target nucleic acid, comprising circularizing and topologically linking an allosterically-regulatable polynucleotide as described above to the target according to the methods described herein.
In another aspect, the invention provides methods for detecting the presence or absence of a target nucleic acid molecule. Such methods comprise contacting a composition suspected of containing the target with an allosterically-regulatable polynucleotide as described above and detecting circularization and complex formation of the polynucleotide with the target, wherein circularization and complex formation is indicative of the presence of the target in the composition, if any. In one embodiment, the target is linked to a solid support, for example, a hybridization membrane. In another embodiment, the allosterically-reulatable regulatable polynucleotide is linked to a solid support. Optionally, a plurality of the polynucleotides may be provided as an array. In some embodiments, detection of the bound and topologically linked polynucleotide is via amplification of the bound polynucleotide, such as, for example, by rolling circle amplification (RCA) as described and exemplified herein. Other suitable amplification procedures are well known in the art, such as, for example, polymerase chain reaction (PCR), RT-PCR, or isothermal amplification methods. The isothermal methods include displacement amplification (Spargo et al., 1996), transcription-mediated amplification (Pastemack et al., 1997), self-sustained sequence replication (3SR) (Mueller et al., 1997), nucleic acid sequence based amplification (NASBA) (Heim et al., 1998), an assay based on the formation of a three-way junction structure (Wharam et al., 2001), ramification amplification (Zhang et al., 2001), loop-mediated amplification (LAMP) (Endo et al., 2004; Nagamine et al., 2002).
In some embodiments, detection is via a detectable label, which may be included on the allosterically-regulatable polynucleotide, the target, or both. Examples of suitable detectable labels are well known in the art, including but not limited to, radioactive, fluorescent, hapten, or enzymatic labels, or labels that comprise members of ligands capable of tight binding, such as biotin-avidin, biotin-streptavidin, antibody-antigen, etc. In other embodiments, detection is via signal amplification methods, including, for example, serial invasive signal amplification reaction (Hall et al., 2000; Olson et al., 2004), branch chain DNA (b-DNA) technology (Wiber, 1997), tyramide signal amplification (TSAD) and catalyzed assisted reported deposition (CARD) (Rapp et al., 1995).
In another aspect, the invention provides a library comprising a plurality of allosterically-regulatable polynucleotides as described above, and methods for preparing such libraries are described and exemplified herein. In various embodiments, libraries of the invention comprise at least one partially randomized sequence in at least one of the target binding sequence, the catalytic domain, and the regulatory sequence.
The invention also provides a method for selection of polynucleotides that are capable of circularizing around and topologically linking to a target nucleic acid molecule. Such methods comprise contacting the target with a plurality of polynucleotides from a library as described above, and amplifying the polynucleotides which become topologically linked to the target. Optionally, multiple rounds of amplification and selection may be performed to increase the specificity of binding of the selected polynucleotides to the target.
In another aspect, the invention provides kits. In one embodiment, the kit comprises an allosterically-regulated polynucleotide as described above. In another embodiment, the kit comprises a library comprising a plurality of allosterically-regulatable polynucleotides as described above.
The invention provides novel allosterically-regulatable polynucleotide molecules that have advantages over currently-existing nucleic acid binding agents. Polynucleotides of the invention can be used, for example, to reduce the efficiency of transcription or translation, for detection and imaging of nucleic acid targets, target validation, or gene function analysis, or as antimicrobial (e.g., antiviral, antibacterial) drugs, or for gene therapy. Polynucleotides of the invention may also be used, for example, as hybridization probes (e.g., for Northern or Southern blots, in situ hybridization, microarrays) with utility in the fields of genomics, biodefense, forensics, microbiology, virology, and oncology. A polynucleotide of the invention includes both a target binding sequence which is capable of binding to a sequence of a target nucleic acid molecule and an ability to circularize that is inhibited in the absence of binding of the polynucleotide to the target molecule. Upon binding of the target binding sequence to the target, a structural rearrangement occurs, allowing circularization of the polynucleotide around the target nucleic acid molecule. As used herein, “circularization” encompasses both covalent and noncovalent interactions that create a circular domain.
In one embodiment, “circularization” involves noncovalent interactions within the polynucleotide that create a “circular” domain encompassing the target binding region. (
In an another embodiment, the polynucleotide of the invention includes a catalytic domain having an ability to induce circularization that is inhibited in the absence of binding of the polynucleotide to the target molecule. Upon binding of the target binding sequence to the target, a conformational change in the polynucleotide allows catalytic action by the catalytic domain resulting in “circularization” of the polynucleotide around the target. (
Previously-described Lassos contain a non-allosterically-regulated hairpin ribozyme (HPR) domain that can spontaneously adopt either a linear or circular conformation. (PCT Application No. WO 99/09045; Australian Patent No. AU756301) In contrast, the polynucleotides of the present invention are allosterically regulated. In the present invention, the efficacy of the Lasso topological linkage to the target and target sequence specificity are enhanced by making the Lasso circularization target-dependent using allosteric regulation.
Allosteric regulation of ribozymes, based on competition between a “sensor” sequence and an external effector sequence supplied by either a synthetic oligonucleotide or a target sequence, has been previously described (Porta & Lizardi, 1995; George et al., 1998; Robertson & Ellington, 2000; Soukup & Breaker, 2000; Warashina et al., 2000; Kazakov, 2001; Burke et al., 2002; Komatsu et al., 2002; Wang et al., 2002; Silverman, 2003). The sensor sequence is designed to be partially complementary to a sequence on or near the ribozyme so as to create in interaction that interferes with the normal functioning of the ribozyme in the absence of the effector. The sensor sequence is designed so that it has higher affinity to the complementary effector sequence than to a functionally important ribozyme sequence. Upon binding of the sensor to the effector, the ribozyme catalytic domain becomes unmasked, and, therefore, active either as a nuclease or ligase or both. Even without extensive rational design, a more than 250-fold rate enhancement in the effector-activated hammerhead ribozyme reaction has been previously observed (Burke et al., 2002; Silverman, 2003). The limiting extent of activation is likely to be proportional to the ratio of relative stabilities of the sensor-effector duplex with the folded core to the sensor-ribozyme complex. It should be possible to further optimize this ratio through rational design or exploiting evolutionary optimization through the in vitro selection (SELEX) (Burke et al., 2002).
General Techniques
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, such as: “Molecular Cloning: A Laboratory Manual,” vol. 1-3, third edition (Sambrook et al., 2001); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987); “PCR Cloning Protocols,” (Yuan and Janes, eds., 2002, Humana Press).
Polynucleotides of the Invention
Polynucleotides of the invention, including those termed “Lassos” herein, specifically bind to a target nucleic acid molecule and circularize around the target. Circularization is dependent on binding of the polynucleotide to a sequence of the target. The polynucleotide contains both a target binding sequence and a means of creating a circular domain that encompasses the target binding sequence. In some embodiments, the means of circularization involves action by a nucleic acid catalytic domain. The catalytic domain is unable to cause circularization in the absence of binding of the target binding sequence to the target. Upon binding of the polynucleotide to the target, the circularization can proceed. Catalytic activity of the catalytic domain serves to circularize the polynucleotide around the target, forming a topological linkage of the polynucleotide with the target. In other embodiments, the means of “circularization” includes formation of noncovalent interactions that create a circular domain around the target, forming a “topological linkage” of the polynucleotide to the target.
As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length and any three-dimensional structure and single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Any type of modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2′-O—Me, phosphorothioates, etc.). Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin. The-term polynucleotide also includes peptide nucleic acids (PNA). Polynucleotides may be naturally occurring or non-naturally occurring. The terms “polynucleotide” and “nucleic acid” and “oligonucleotide” as used herein are used interchangeably. Polynucleotides of the invention may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof. A sequence of nucleotides may be interrupted by non-nucleotide components. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Polynucleotides may be linear or circular or comprise a combination of linear and circular portions. The terms “polynucleotide” and “nucleic acid” and “oligonucleotide” as used herein are used interchangeably.
Allosterically-regulatable polynucleotides of the invention include a target binding sequence and may include a catalytic domain. As used herein, “target binding sequence” or “antisense sequence” refers to a sequence that is at least partially complementary and capable of binding to a sequence of a target nucleic acid. In some embodiments, the target binding sequence is about 10 to about 30, often about 20, base pairs in length. The target binding sequence may be fully complementary to the target sequence, or there may be one or more mismatches between the binding sequence and the target, so long as binding is sequence specific and tight enough to disrupt alternative interactions that prevent circularization in the absence of target binding. The target binding sequence generally comprises a sequence that is at least “substantially complementary” to a sequence of the target molecule, meaning a sequence that is sufficiently complementary to allow hybridization therebetween via normal base pair binding. Substantially complementary sequences may be fully complementary or may have one or more mismatch(es). Either or both of the target binding sequence and the target may comprise DNA, RNA, or both, and/or analogs or modified forms thereof, and/or modified internucleotide linkages.
As used herein, “catalytic domain” refers to a nucleic acid sequence that is capable of catalyzing a reaction, for example, ligation between nucleotides or cleavage and subsequent ligation of a nucleic acid sequence. In one embodiment, the catalytic domain is capable of catalyzing a ligation reaction between 5′ and 3′ ends of a polynucleotide molecule of the invention to circularize and topologically link the polynucleotide to the target. An example of such a catalytic domain is the catalytic domain of the hairpin ribozyme, as shown in
The “target,” “target sequence,” or “target nucleic acid” as used herein is a polynucleotide comprising a sequence of interest. The target may comprise DNA, RNA, or both, and/or analogs or modified forms thereof, and/or modified internucleotide linkages. In some embodiments, the target is mRNA, genomic DNA, cDNA, cRNA, viral RNA, ribosomal RNA, non-coding RNA, a viral RNA-DNA replication intermediate, or an RNA-protein complex. Allosterically-regulatable polynucleotides of the invention become topologically linked to the target by one or more of a variety of mechanisms described herein, regardless of the structure of the target nucleic acid. The target nucleic acid may be linear, circular, or may comprise both linear and circular portion(s), or may take any other form that allows topological linkage of a polynucleotide of the invention thereto.
As used herein, “topological linkage” refers to intertwining of a circularized polynucleotide of the invention with the target nucleic acid molecule (see
Topological linkage of a polynucleotide of the invention is allosterically regulatable, with circularization dependent on target binding. Circularization is blocked in the absence of target binding. In one embodiment, circularization is inhibited by a “regulatory” (also termed “inhibitory” or “inhibitor” herein) nucleic acid which binds to at least a portion of the target binding sequence, thereby preventing circularization of the polynucleotide when it is not bound to the target. In one embodiment, the regulatory sequence is a sequence of the allosterically-regulatable polynucleotide, either internal to the polynucleotide or at one or both of the ends. In another embodiment, the regulatory sequence is on a different nucleic acid than the allosterically-regulatable polynucleotide. In one embodiment, the regulatory nucleic acid sequence comprises a sequence that is at least partially complementary, often substantially complementary, sometimes fully complementary to the target binding sequence. The regulatory element may include mismatches and still maintain high fidelity of binding to the intended target. The regulatory element-target binding sequence binding needs only to be strong enough to block circularization in the absence of binding of the target binding sequence to sequences of the target nucleic acid. Binding between the regulatory sequence and the target binding sequence improves specificity of binding of the polynucleotide to the target through competition between the regulatory sequence and the sequence of the target to which the target binding sequence binds. In embodiments in which circularization is dependent on catalytic activity, binding of the target binding sequence to the target displaces the regulatory sequence, which allows catalytic action by the catalytic domain, resulting in circularization of the polynucleotide around the target.
The invention also provides a complex comprising an allosterically-regulatable polynucleotide as described above circularized around and topologically linked to a target molecule. Often, formamide is included in the reaction mixture for complex formation. In various embodiments, about 5, 10, 15, or 20% formamide is used. Formamide has been reported to provide a stronger correlation between in vitro and in vivo efficacy of ribozymes (Crisell et al., 1993; Kisich et al., 1997; Sullivan et al., 2002). Presence of the complex may reduce efficiency of transcription and/or translation from the target nucleic acid.
Methods for Topologically Linking a Polynucleotide Lasso to a Target Molecule
The invention provides methods for circularizing an allosterically-regulatable polynucleotide molecule as described above around a target molecule, forming a topological linkage. A method of the invention includes contacting a composition containing the target molecule with a polynucleotide that comprises a target binding sequence, wherein binding of the target binding sequence to the target allows circularization and topological linkage of the target to proceed.
In one embodiment, the polynucleotide comprises a target binding sequence and a catalytic domain capable of catalytic action, and binding of the target binding sequence to the target allows catalytic action to proceed, resulting in circularization and topological linkage of the polynucleotide to the target. In the absence of target binding, catalysis does not occur, preventing or significantly reducing circularization and topological linkage to the target. Often, inhibition of catalysis is effected by an regulatory sequence as described above. In one embodiment, the catalytic activity is a ligase activity, causing ligation between the 5′ and 3′ ends of the polynucleotide to form a circular structure around the target. In another embodiment, the catalytic activity is a ligase activity, causing ligation between the 5′ end and a 2′ hydroxyl group of an internal nucleotide residue to form a lariat shaped structure whose circular part is intertwined with the target. 2′-5′ ligation allows potential labeling at the free 3′ end of the polynucleotide, which may be used for detection of topologically linked polynucleotides and targets. As used herein, “ligation” refers to the formation of a phosphodiester bond between a hydroxyl group of one nucleotide and a phosphate group of another nucleotide, e.g., the 3′-OH or 2′-OH of one nucleotide and a 5′-phosphate group of another nucleotide, such that there are no intervening nucleotides between the nucleotides that have been joined by ligation.
In another embodiment, the polynucleotide comprises a target binding sequence and binding of the target binding sequence to the target causes circularization to proceed via structural rearrangement within the polynucleotide that creates a circular domain encompassing the target binding sequence, circularizing the polynucleotide around the target and forming a topological linkage.
The invention also provides methods for reducing the efficiency of transcription and/or translation from a target, or inhibiting or redirecting splicing, comprising topologically linking an allosterically regulated polynucleotide to the target as described above. Transcription and/or translation may be partially reduced or fully eliminated. Reduction of transcription or translation may be detected by methods that are well known in the art including, but not limited to, Northern or Southern blots or RT-PCR for transcription or Western blots or ELISA (enzyme-linked immunorbant assay) for translation. In various embodiments, transcription or translation is reduced at least about 10, 20, 30, 40, 50, 60, 70, 80, or 90%, or is fully eliminated, depending on specific factors such as the accessibility of the target site, the efficiency of binding and regulation of the polynucleotide and other influences, due to formation of a complex comprising a topologically linked polynucleotide of the invention to the target.
In principle, allosterically regulated polynucleotide-target complexes can disable target RNA by three distinct processes: physically blocking its functional sequences, disruption of functionally active structures, and induction of its degradation. Within these broadly defined processes, different mechanisms are possible. These mechanisms include but are not limited to translation arrest, prevention of RNA processing, regulation of alternative splicing or nuclear retention of target transcripts, depending on the design of the allosterically regulatable polynucleotide, the target sequence chosen, or the selection procedure used to identify effective allosterically regulated circularizable polynucleotides from combinatorial libraries. Allosterically regulatable polynucleotides of the invention can be delivered to cellular targets either directly in liposomal complexes or through expression in situ from plasmids or viral vectors.
Methzods for Detecting Presence or Absence of a Target Nucleic Acid Molecule
The invention provides methods for detecting the presence or absence of a target nucleic acid molecule. Methods of detection include contacting a composition suspected of containing a target molecule with an allosterically-regulatable polynucleotide as described above, and detecting circularized polynucleotide topologically linked to the target, wherein presence of the circularized polynucleotide indicates presence of the target molecule in the composition, if any, and absence of the circularized polynucleotide indicates absence of the target molecule.
In one embodiment, the target molecule is associated with or bound to a solid support, e.g., a hybridization membrane, e.g., nitrocellulose or nylon (dot blots, Northern blots, Southern blots), modified glass, silicon or gold surfaces (microarrays), modified magnetic or glass beads (affinity capture).
In another embodiment, the allosterically-regulatable polynucleotide molecule is associated with or bound to a solid support. For example, the polynucleotide may be comprised within an array. “Microarray” and “array,” as used interchangeably herein, comprise a surface with an array, preferably ordered array, of putative binding (e.g., by hybridization) sites for a biochemical sample (target) which often has undetermined characteristics. In a preferred embodiment, a microarray refers to an assembly of distinct allosterically-regulatable polynucleotides as described above immobilized at defined positions on a substrate. Arrays are formed on substrates fabricated with materials such as paper, glass, plastic (e.g., polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose, silicon, optical fiber or any other suitable solid or semi-solid support, and configured in a planar (e.g., glass plates, silicon chips) or three-dimensional (e.g., gels, pins, fibers, beads, particles, microtiter wells, capillaries) configuration. Probes forming the arrays may be attached to the substrate by any number of ways including (i) in situ synthesis (e.g., high-density oligonucleotide arrays) using photolithographic techniques (see, Fodor et al., Science (1991), 251:767-773; Pease et al., Proc. Natl. Acad. Sci. U.S.A. (1994), 91:5022-5026; Lockhart et al., Nature Biotechnology (1996), 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270); (ii) spotting/printing at medium to low-density (e.g., cDNA probes) on glass, nylon or nitrocellulose (Schena et al, Science (1995), 270:467-470, DeRisi et al, Nature Genetics (1996), 14:457-460; Shalon et al., Genome Res. (1996), 6:639-645; and Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1995), 93:10539-11286); (iii) by masking (Naskos and Southern, Nuc. Acids. Res. (1992), 20:1679-1684) and (iv) by dot-blotting on a nylon or nitrocellulose hybridization membrane (see, e.g., Sambrook et al., Eds., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.)). Polynucleotides may also be noncovalently immobilized on the substrate by hybridization to anchors, by means of magnetic beads, or in a fluid phase such as in microtiter wells or capillaries.
Detection of topologically linked polynucleotides may be by any of a number of methods that are well known in the art, including, for example, detecting a, label on either the allosterically-regulatable polynucleotide molecule and/or the target nucleic acid molecule. Detectable labels include, for example, radioisotopes (e.g., 3H, 35S, =P, 33P, 125I, or 14C), fluorescent dyes (e.g., fluorescein isothiocyanate, Cy3, Cy5, Texas red, rhodamine, green fluorescent protein, and the like), enzymes (e.g., LacZ, horseradish peroxidase, alkaline phosphatase, luciferase), digoxigenin, and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and members of binding pairs such as biotin-avidin, biotin-strepavidin, antibody-antigen, etc., wherein one member of the binding pair is labeled and is detected by binding to the other member of the binding pair. Detection of any of the above labels may be qualitative and/or quantitative.
Detection of topologically linked polynucleotides may also be via amplification of the bound polynucleotide molecule. Any amplification method known in the art may be used, so long as it yields a detectable amount of amplified product. Amplification may include, for example, by rolling circle amplification and/or polymerase chain reaction. As used herein, “amplification” refers to the process of producing multiple copies of a desired nucleic acid sequence or its complement. “Multiple copies” means at least two copies. A “copy” does not necessarily have to have perfect complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations, for example introduced via a primer, and/or sequence errors that occur during amplification. “Rolling circle amplification” refers to an amplification process whereby circularized polynucleotide molecules of the invention that are topologically linked to a target are amplified. Circularized polynucleotide molecules of the invention that are topologically linked to a target can be isolated by affinity (hybridization) capture of the target and subsequent synthesis of a long, single-stranded copy of the circular polynucleotide by a polymerase, typically reverse transcriptase, moving around the circle multiple times in a rolling circle scheme. An example of rolling circle amplification process for polynucleotides of the invention is provided in Example 13 below. Other suitable amplification procedures are well known in the art, such as, for example, polymerase chain reaction (PCR), RT-PCR, or isothermal amplification methods. The isothermal methods include displacement amplification (Spargo et al., 1996), transcription-mediated amplification (Pastemack et al., 1997), self-sustained sequence replication (3 SR) (Mueller et al., 1997), nucleic acid sequence based amplification (NASBA) (Heim et al., 1998), an assay based on the formation of a three-way junction structure (Wharam et al., 2001), ramification amplification (Zhang et al., 2001), loop-mediated amplification (LAMP) (Endo et al., 2004; Nagamine et al., 2002).
Methods of Preparation of Allosterically-Regulatable Polynucleotide Lasso Molecules
Allosterically-regulatable polynucleotides as described above may be prepared by any method known in the art for preparation of polynucleotide molecules. For example, the polynucleotides may be prepared synthetically or expressed from an expression vector.
Polynucleotides of the invention may be prepared synthetically using methods that are well known to those of skill in the art, including, for example, direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99, the phosphodiester method of Brown et al.(1979) Meth. Enzymol. 68: 109-151, the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862, or the solid support method of U.S. Pat. No. 4,458,066. Synthetic methods may be used to produce polynucleotides that contain deoxyribonucleotides, ribonucleotides, and/or modified forms or analogs thereof.
Polynucleotides of the invention may also be prepared via transcription from an expression vector. Transcription may be in vitro or may occur in vivo in an appropriate host cell. A nucleic acid encoding an allosterically-regulatable polynucleotide of the invention can be incorporated into a recombinant expression vector in a form suitable for in vitro or in vivo expression. As used herein, an “expression vector” is a nucleic acid which includes appropriate sequences to facilitate expression (e.g., replication or transcription) of an incorporated polynucleotide of interest. For in vivo expression, an expression vector can be introduced into an appropriate host cell. An expression vector may include transcriptional regulatory elements such as promoters, e.g., the T7 promoter, and/or enhancers and/or other expression control elements (e.g., polyadenylation signals). Such sequences are known to those skilled in the art (see, e.g., Goeddel (1990) Gene Expression Technology: Meth. Enzymol. 185, Academic Press, San Diego, Calif.; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif.; Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, etc.). In some embodiments, a recombinant expression vector is a plasmid or cosmid. In other embodiments, the expression vector is a virus, or portion thereof, that allows for expression of a nucleic acid introduced into the viral nucleic acid. For example, replication defective retroviruses, adenoviruses and adeno-associated viruses can be used. Preferred in vitro expression systems include, for example, run-off transcription using bacterial, T7, SP6, and T3 RNA polymerase from appropriate templates, including single-stranded DNA templates (linear and circular), double-stranded DNA templates, and-plasmid vectors. Preferred in vivo expression systems include, for example, double-stranded DNA templates and plasmid vectors having Pol II or Pol III RNA polymerase promoters, or viral, e.g., lentiviral, vectors.
Viral expression vectors may be derived from bacteriophage, including all DNA and RNA phage (e.g., cosmids), or eukaryotic viruses, such as baculoviruses and retroviruses, adenoviruses and adeno-associated viruses, Herpes viruses, Vaccinia viruses and all single-stranded, double-stranded, and partially double-stranded DNA viruses, all positive and negative stranded RNA viruses, and replication defective retroviruses. Another example of an expression vector is a yeast artificial chromosome (YAC), which contains both a centromere and two telomeres, allowing YACs to replicate as small linear chromosomes. A number of suitable expression systems are commercially available and can be modified to produce the vectors of this invention. Illustrative expression systems include, but are not limited to baculovirus expression vectors (see, e.g., O'Reilly et al. (1992) Baculovirus Expression Vectors: A Laboratory Manual, Stockton Press) for expression in insect (e.g. SF9) cells, a wide variety of expression vectors for mammalian cells (see, e.g., pCMV-Script® Vector, pCMV-Tag1, from Stratagene), vectors for yeast (see, e.g., pYepSec1, Baldari et al. (1987) EMBO J. 6: 229-234, pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and the like), prokaryotic vectors (see, e.g., arabinose-regulated promoter (Invitrogen pBAD Vector), T7 Expression Systems Novagen, Promega, Stratagene), Trc/Tac Promoter Systems (Clontech, Invitrogen, Kodak, Life Technologies, MBI Fermentas, New England BioLabs, Pharmacia Biotech, Promega), PL Promoters (Invitrogen pLEX and pTrxFus Vectors), Lambda PR Promoter (Pharmacia pRIT2T Vector), Phage T5 Promoter (QIAGEN), teta Promoter (Biometra pASK75 Vector), and the like.
Allosterically-regulatable polynucleotides of the invention can be expressed in a host cell. As used herein, the term “host cell” is intended to include any cell or cell line into which a recombinant expression vector for production of an allosterically-regulatable polynucleotide, as described above, may be transfected. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected or transformed in vivo with an expression vector as described above.
Suitable host cells include, but are not limited to, to algal cells, bacterial cells (e.g. E. coli), yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis,. H. polymorpha, (see, e.g., Fleer (1992) Curr. Opin. Biotech. 3(5): 486-496), fungal cells, plant cells (e,-g. Arabidopsis), invertebrate cells (e.g. insect cells such as SF9 cells, and the like), and vertebrate cells including mammalian cells. Non-limiting examples of mammalian cell lines which can be used include CHO cells (Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220), 293 cells (Graham et al. (1977) J. Gen. Virol. 36: 59), or myeloma cells like (e.g., SP2 or NS0, see Galfre and Milstein (1981) Meth. Enzymol. 73(B):3-46). In one embodiment, the expression system includes a baculovirus vector expressed in an insect host cell.
An expression vector encoding a allosterically-regulatable polynucleotide of the invention can be transfected into a host cell using standard techniques. “Transfection” or “transformation” refers to the insertion of an exogenous polynucleotide into a host cell. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome. The term “transfecting” or “transfection” is intended to encompass all conventional techniques for introducing nucleic acid into host cells. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation and microinjection. Suitable methods for transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, and other laboratory textbooks. Nucleic acid can also be transferred into cells via a delivery mechanism suitable for introduction of nucleic acid into cells in vivo, such as via a retroviral vector (see e.g., Ferry et al. (1991) Proc. Natl. Acad Sci., USA, 88: 8377-8381; and Kay et al. (1992) Human Gene Therapy 3: 641-647), an adenoviral vector (see, e.g., Rosenfeld (1992) Cell 68: 143-155; and Herz and Gerard (1993) Proc. Natl. Acad. Sci., USA, 90:2812-2816), receptor-mediated DNA uptake (see e.g., Wu, and Wu (1988) J. Biol. Chem. 263: 14621; Wilson et al. (1992) J. Biol. Chem. 267: 963-967; and U.S. Pat. No. 5,166,320), direct injection of DNA (see, e.g., Acsadi et al. (1991) Nature 332: 815-818; and Wolff et al. (1990) Science 247:1465-1468) or particle bombardment (biolistics) (see e.g., Cheng et al. (1993) Proc. Natl. Acad. Sci., USA, 90:4455-4459; and Zelenin et al. (1993) FEBS Letts. 315: 29-32).
Certain vectors integrate into host cells at a low frequency. In order to identify these integrants, in some embodiments a gene that contains a selectable marker (e.g., drug resistance) is introduced into the host cells along with the nucleic acid of interest. Examples of selectable markers include those which confer resistance to certain drugs, such as G418 and hygromycin. Selectable markers can be introduced on a separate vector from the nucleic acid of interest or on the same vector. Transfected host cells can then be identified by selecting for cells using the selectable marker. For example, if the selectable marker encodes a gene conferring neomycin resistance, host cells which have taken up nucleic acid can be identified by their growth in the presence of G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die.
In one embodiment, an allosterically-regulatable polynucleotide of the invention is prepared by expression by RNA polymerase II or III in the nucleus of a host cell.
Libraries
The invention also provides libraries comprising a plurality of allosterically-regulatable polynucleotides as described above, each of which comprises a target binding sequence. Each polynucleotide may also include regulatory sequence(s) which prevent circularization of the polynucleotide around the target in the absence of binding of the target binding sequence to the target. In one embodiment, each polynucleotide comprises a target binding sequence and a catalytic domain which is capable of catalytic activity to circularize the polynucleotide around the target upon binding of the target binding sequence to the target. In various embodiments, the target binding sequence, the regulatory sequence, and/or the catalytic domain are at least partially randomized.
A variety of methods for preparation of libraries of polynucleotides are well known in the art. For example, polynucleotide libraries having randomized sequence inserts may be prepared either synthetically (either as a mixture of rationally selected sequences or partially or fully random sequences, or may be derived from directed (gene- or genome-specific) libraries. Directed libraries may be prepared by nuclease digestion, e.g., using a combination of Exonuclease III/Mung bean/BsmFI-Bbs restriction (type IIS) nucleases (Pierce and Ruffner (1998) Nucleic Acids Res. 26:5093-101; WO 99/50457), or DNase I/MmeI (Shirane et al. (2004) Nat. Genet. 36:190-196), or by consecutive digestion using a mixture of HinpI-BsaHI-AciI-HpaHI-HpyCHIV-TaqaI restriction endonucleases in combination with MmeI nuclease (Sen et al. (2004) Nat. Genet. 36:183-189). Alternative approaches for preparation of directed libraries include PCR amplification of hemi-random oligonucleotides that are selected based on either direct hybridization to immobilized DNA target (WO 00/43538; Bruckner et al. (2002) Biotechniques 33:874-882) or template-dependent ligation on DNA/RNA target templates (WO 03/100100A1).
The invention also provides a method for selection of polynucleotides that are capable of circularizing and-topologically linking to a target nucleic acid molecule, comprising contacting a target molecule with allosterically-regulatable polynucleotides from a library as described above, and amplifying the polynucleotides which become topologically linked to the target.
A novel selection approach described herein, which starts with randomized libraries of Lassos, provides simultaneous selection of both accessible target sites and optimal design of the Lasso so that circularization is dependent on prior hybridization to the target. Individual members of a Lasso library may differ from one another as follows. They may contain antisense sequences complementary to different segments of the target. They may also differ in the sequence of the circularizing moieties, for example partially randomized derivatives of naturally-occuring or naturally-existing, e.g., hairpin ribozyme, or catalytic nucleic acids derived by in vitro selection. The antisense sequences may constitute either fully random or “directed,” gene-specific libraries of antisense sequences. Circularization of Lassos is may be regulated by introduction of an regulatory element, optionally also containing partially randomized sequences.
In one embodiment, any of loops 1-3 in the HPR domain (see
Below is a partial list of possible combinations of partial/filly randomized sequences that can be used to generate an initial pool of Lassos for selection and amplification by RCA-PCR:
Selection with an increased number of randomized nucleotides in each of the above-mentioned regions can result in an unpredicted, but optimally effective molecular mechanism of allosteric regulation of the Lasso. In each case, the pool of Lassos is incubated with target and Lassos that can circularize in a target-dependent manner are selectively amplified by RCA-RT-PCR as described above (
In addition, sequences in the catalytic domain (i.e., catalytically essential residues) could be further altered (rationally or using SELEX) in a way to improve the efficacy of the cleavage and ligation reactions.
Kits
The invention also provides kits that include one or more allosterically-regulatable polynucleotides or libraries as described above. Kits of the invention include separately or in combination allosterically-regulatable polynucleotides, libraries containing such polynucleotides, reagents such as buffers, expression vectors, host cells, growth medium, reagents for detection and/or amplification of topologically-linked polynucleotide-target complexes, and/or reagents for preparing libraries or arrays.
Each reagent is supplied in a solid form or liquid buffer that is suitable for inventory storage, and later for exchange or addition into a reaction or culture medium. Suitable packaging is provided. As used herein, “packaging” refers to a solid matrix or material customarily used in a system and capable of holding within fixed limits one or more polynucleotides or libraries of the invention or one or more reagent components for use with the polynucleotides, libraries, and/or methods of the invention. Such materials include glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic, and plastic-foil laminated envelopes and the like.
In addition, the kits optionally include instructional materials providing directions (i.e., protocols) for the practice of the methods of this invention. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to Internet sites that provide such instructional materials.
Exemplification of the Invention
The invention provides design and methods for the preparation of allosterically regulated Lassos, capable of rapid, sequence-specific hybridization to nucleic acid targets and target-dependent circularization creating a strong topological link between Lasso and target. RNA Lassos containing a non-regulated hairpin ribozyme (HPR) domain that can spontaneously adopt either a linear or circular conformation have been previously described (Johnston et al., 1998, 2003). To improve the efficacy and specificity of Lasso binding to and circularization around the target, we have developed a method to make the Lasso circularize only after it binds to the target using a unique version of allosteric regulation. As proof of principle, we performed the following experiments, which are described in greater detail in the Examples below.
First, we modified the originally used hairpin ribozyme core sequence described in Feldstein & Bruening, 1993, to make it more efficient in self-processing (including both cleavage and ligation steps) according to Esteban et al. (1997). Second, we chose another antisense sequence than the one used in ATR1. This new antisense sequence corresponds to the TNFα mRNA site shown to be accessible in vivo in experiments with hammerhead ribozyme (Sioud et al., 1992, 1994, 1996; Kisich et al., 1997). Third, to make the Lasso self-processing and ligation target-dependent, we employed a scheme of allosteric regulation similar to the “TRAP”-like mechanism previously described for the cleavage reaction catalyzed by the hammerhead ribozyme (Porta & Lizardi, 1995; George et al., 1998; Burke et al., 2002. Herein, for the first time, we employed allosteric regulation for regulating the cleavage and ligation activity of the hairpin ribozyme. As with the unregulated Lasso, we used the ribozyme not for cleaving the target but rather to circularize the ribozyme around the target.
We achieved allosteric regulation by introducing complementarity between the Lasso's antisense sequence (sensor element) and another sequence of the Lasso (regulatory or inhibitory element) so as to form a regulatory complex-that prevents circularization in the absence of hybridization with target RNA. The region of internal pairing on the antisense (sensor) sequence, was chosen so that the antisense sequence has higher affinity to the complementary target sequence, which serves as the effector than to the complementary regulatory sequence. Upon binding of the antisense (sensor) sequence to the target (effector) sequence, the regulatory complex was disrupted, allowing self-processing of the Lasso ends and their ligation around the target to proceed.
As an example of an allosterically regulated Lasso, we designed the sensor-antisense sequence to be complementary to the ‘regulatory’ element, comprising hairpin ribozyme 3′ end substrate sequence (5-nt long) extended by a few nucleotides (typically, 0 to 5 nt) into the Lasso loop sequence (
A series of allosterically regulated Lassos was designed and synthesized to target the 229-249 region of murine TNFα in the long TNF2 transcript (709 nt) as well as with the short TNF-20 (20 nt long) synthetic RNA. These Lasso derivatives (ALR229-5, 229-6, 229-7, 229-8, 229-9 and 229-10) differed in the length of the regulatory elements (i.e., having 5, 6, 7, 8, 9 and 10 nucleotides complementary to the Lasso antisense domain) (
As expected, all initial Lasso transcripts underwent self-cleavage at their 5′ ends during transcription. The Lassos having the shortest regulatory sequence, ALR229-5 and ALR229-6, also processed their 3′ ends and underwent circularization, whereas 3′ end cleavage of Lassos ALR229-7 through ALR229-10 was inhibited, indicating allosteric regulation (
By displacing Lassos bound to TNF2 using highly denaturing conditions (60% formamide/10 mM EDTA, 95° C.), we detected the circularization of ALR229-7-8-9 Lassos induced by target binding albeit not in great yield (
Allosterically-regulated-Lasso RNAs.(e.g. 229-7(0)) are active under a wide variety of buffer conditions. When target-binding and target-dependent circularization was tested in buffer conditions considered to be more physiologically relevant (20 mM HEPES, pH 7.3, 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM CaCl2) than our standard buffer conditions (50 mM Tris-Cl pH 8, 10 mM MgCl2, 20% formamide), 229-7(0) was capable of target-dependent circularization and bound to target TNF2 (
Allosterically-regulated Lassos were rationally designed to bind to other target sites. For example, we designed allosterically-regulated Lassos that bound to the target TNF 562-583. We designed several Lassos with different lengths of the regulatory sequences (ranged between 7 and 10 nt) identified as ALR562-1 through 562-4) (
To prove that Lasso circularization upon binding to the RNA target does, in fact, result in topological link formation, we analyzed binding of the allosterically regulated Lasso 229-7(0) (
Single and multiple mutations were introduced into MuTNF targets by site-directed mutagenesis to demonstrate that allosterically-regulated Lassos confer higher sequence-specificity in comparison to non-allosterically regulated Lassos. When mismatches to target sequences occurred at nucleotide positions complemented by the regulatory element, little or no complex formation was detected for the allosterically regulated Lasso 229-7(0). However, 229-5, which is not allosterically regulated, formed complexes with the mutated target RNAs that were stable under denaturing gel electrophoresis (
To both select and amplify circular molecules from a pool of RNA that contains both linear and circular RNAs, we developed a procedure involving RCA (rolling circle amplification) and RT-PCR steps (
In this scheme, primer 1, which is complementary to the 5′ end of the 5′-processed Lasso, hybridizes across the active site of the HPR domain, thus inhibiting its catalytic activity and preventing further processing of the Lasso during subsequent manipulations even in the presence of Mg2+ ions. In the reverse transcription (RT) reaction, primer 1 selectively extends only circular Lassos, yielding single-stranded DNA multimers of the Lasso sequence (via RCA). Two additional primers (primer 2 and primer 3) are then used to amplify the RCA product by PCR and to add the T7 promoter sequence at the Lasso's 5′ end so that the products may be transcribed in vitro. As proof of principle, we were able to selectively amplify the circular form of Lasso 229-5 using this technique. In
We carried out a selection using the scheme shown in
Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope of the invention. Therefore, the description should not-be construed as limiting the scope of the invention, which is delineated by the appended claims.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their, entireties for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.
The following examples are provided to illustrate but not limit the present invention.
Construction of DNA Template for in vitro Transcription of Lassos
A series of six Lassos containing from 5-10 internal base pairs to the antisense sequence (nt 229-248 of murine TNFα RNA) were constructed (229-5,229-6,229-7, 229-8, 229-9,229-10), as shown in
Two partially complementary overlapping oligonucleotides were used for 229-5 through 229-10 as follows (shown in 5′-3′ direction):
The oligonucleotides were annealed at 80° C. for 5 minutes and slowly cooled to room temperature over the course of an hour. The oligonucleotides were filled in by Klenow extension to create a double-stranded template. Primers used to amplify this sequence using PCR and to add a T7 promoter sequence were as follows:
PCR products were purified on 1.6% agarose gel. These gel-purified fragments were used as templates for in vitro run off transcription by T7 RNA Polymerase.
In Vitro Transcription of Lassos
Lassos were in vitro transcribed using T7 RNA polymerase (Promega) for 3-5 hours at 37° C. using [32P-α]CTP in the transcription mixture. Transcripts were desalted over a G50 micro-spin column (Amersham) and were stored at −20° C. until further use.
Assays were performed using internally-radiolabeled Lassos, incubated either alone or with an excess of TNF2 target RNA (cold) at 37° C. for 120 minutes in one of three buffers: (i) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2; (ii) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20% formamide volume/volume; (iii) 20 mM HEPES, pH 7.3, 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM CaCl2. Reactions were quenched with an equal volume of loading buffer containing 90% formamide, 10 MM EDTA, 0.01% bromophenol blue, 0.01% xylene cyanol. Samples were analyzed on 6% PAGE/8M urea/0.5× TBE gels and were electrophoresed at 11 Watts for approximately two hours. Gels were dried and either directly scanned by phosphorimager or exposed to X-ray film.
Lassos 229-5 and 229-6 (five and six base pair regulatory sequences, respectively) were able to fully self-process both 5′ and 3′ ends as evidenced by linear and circular gel bands (
Lassos as described in Example 1 were tested in target binding assays as described in Example 2 in buffer containing 20% formamide. As the length of the regulatory sequence was increased, the efficiency of target binding decreased, while the extent of target-independent processing was reduced (
Lassos 229-5-6-7-8-9-10 were transcribed from DNA templates and tested in binding experiments with the long TNF2 target as well as with the short 20 nt synthetic TNF-20 RNA (Dharmacon) comprising just 20-nt of target TNF sequence (
Lasso transcripts (pre-Lassos) underwent self-cleavage at their 5′-ends during transcription, whereas the cleavage of their 3′-ends was inhibited (see
We showed that the ALR229-5-6-7-8 Lassos bound the long target more strongly and more efficiently than the short target, and also that the Lasso-TNF2 complexes were more stable than Lasso-TNF-20 under the conditions of denaturing PAGE (
Lasso-target complexes were formed and quenched as described in Example 2. To test for target-dependent circularization, half of the Lasso-target complex reaction was heated in loading buffer for 2 minutes at 90° C. and then placed immediately on ice to prevent complex re-hybridization prior to loading on a denaturing gel. Lasso incubated without target RNA, undissociated complex and dissociated complex were loaded in adjacent lanes on 6% denaturing gel. Lasso species (dissociated) were compared to Lasso species present before incubation with target RNA to assess the extent of target-dependent circularization.
Complexes were formed with Lassos 229-5 through 229-10 without formamide present in the incubation buffer. Lassos 229-7, 229-8, and 229-9 show an accumulation of circular Lasso species that was not present when the Lasso was incubated without target. 229-5 and 229-6 contained some circular species before incubation with target and did not show allosteric regulation. After heat treatment, circular form remained, but not more than was originally present. 229-10 did not form complex with target RNA under these conditions. (
A series of Lassos were designed with altered 3′ ends to improve the ability of allosterically regulated Lasso 229-7 to circularize (229-7(0-5)) (
The rationally designed and tested RNA Lasso 229-7(0) has partially self-complementary antisense domains, and was demonstrated to have target-dependent circularization ability with respect to a pre-selected accessible site on TNFα RNA. When Lasso 229-7(0) was incubated alone, only half-processed and some fully-processed linear species were observed (
Lasso 229-7(0) was shown to be capable of target-dependent circularization under buffer conditions considered to be more physiologically relevant (20 mM HEPES, pH 7.3, 140 mM KCl, 10 mM NaCl, 1 mM MgCl2, 1 mM CaCl2) than standard assay buffer conditions (50 mM Tris-Cl pH 8, 10 mM MgCl2, 20% formamide). Under these conditions, Lassos 229-5b and 229-7(0) bound efficiently to target TNF2 and showed similar amounts of circularization after incubation at 37° C. for 120 minutes and subsequent complex displacement by 95° C. treatment as was observed for the standard buffer (
A series of allosterically regulated Lassos was designed to target the TNF 562-583 sequence in the coding region. The regulatory sequences in these Lassos ranged between seven and ten base pairs (
A 120 nt circular RNA target containing the TNF 229-248 nt site was prepared using the strategy described by Beaudry and Perreault (1995). 32P-labeled Lasso 229-7(0) was incubated with linear and circular targets, respectively, under conditions where Lassos can (i.e., in the presence of Mg2+) (
The identity of each complex was assigned by analyzing the products of dissociation after incubating the complexes at 95° C. for 2 min followed by quenching on ice prior to loading on the gel (see
A series of mutated TNF2 targets were synthesized (Stratagene Quick Change mutagenesis kit) with mismatches to the 229-7(0) antisense sequence as shown in
To select and amplify only circular molecules from a pool of Lasso RNA that contains both linear and circular species, we developed a scheme that uses RCA (rolling circle amplification) in an RT-PCR reaction (shown schematically in
In this scheme, a primer used to reverse transcribe only circular molecules (RT primer 1: 5′-GCTTCTCTCGTCATACG-3′ (SEQ ID NO: 10)) was annealed to the unique, complementary sequence near the 5′ end of the Lasso transcript by incubating for 1 min. at 85° C. followed by cooling at room temperature for 5 min. The primer hybridized across the active site of the HPR domain, and prevented further self-processing of the Lasso during subsequent manipulations. In the reverse transcription (RT) reaction, RT primer 1 extended only circular Lasso species selectively, yielding (via RCA) single-stranded DNA multimers of the Lasso sequence. Linear (or unligated) Lassos yielded only a short abortive product, which would not be amplified by PCR in the next step. Several commercially available reverse transcriptases were tested to optimize the procedure. Of those tested, Superscript II (Invitrogen) gave consistent, reliable rolling circle amplification. The RT reaction was carried out for 1 hour with the SuperScript II enzyme according to the manufacturer's protocol. Two additional primers (PCR primer 2: 5′-TAATACGACTCACTATAGGGCAGCCGTC-3′ (SEQ ID NO: 11) and PCR primer 3: 5′-GGTGACACTATGATGCATATGACGAGGAC-3′ (SEQ ID NO: 12)) were then used to amplify the RCA product by PCR and to add the T7 promoter sequence at the 5′-end of the Lasso so that the product could be transcribed by T7 RNA polymerase.
This technique was initially tested on the free Lasso in the absence of its target. We were able to selectively amplify the circular form of a previously characterized Lasso 229-5 (
Preparation of Lasso DNA Cassette Containing a 20N Randomized Target Region
Lasso DNA cassettes encoding a fully randomized 20N target sequence and T7 RNA polymerase promoter were prepared by PCR cloning using the overlapping oligonucleotide scheme described in Example 1 with the exception that the sequences corresponding to 229 antisense were replaced by 20 N randomized bases. Lasso library selection
The 20N Lasso library was transcribed in vitro with T7 RNA polymerase (Ambion) to generate an initial pool of Lassos for in vitro selection (
A mini-library was synthesized of ALR229-5N Lassos (
After the last round of selection, 20 resulting DNA fragments are cloned and sequenced. The obtained sequences are compared to the related sequences of ALR229-5-6-7-8-9-10 are statistically analyzed.
We developed a method for detecting and amplifying only Lassos in their covalently ligated circular form. To detect the circularization of Lasso RNAs, we designed primers for RT-PCR (similar to those shown in
Primer 1 was designed to be complementary to the 5′-end of the 5′-processed Lasso. In the reverse transcription (RT) reaction, Primer I (5′-GCTTCTCTCGTCATACG-3′ (SEQ ID NO: 10)) selectively extended only circular Lassos, yielding single-stranded DNA multimers of the Lasso sequence (rolling circle amplification, RCA). Two additional primers (Primer 2 (5′-TAATACGACTCACTATAGGGCAGCCGTC-3′ (SEQ ID NO: 11)) and Primer 3 (5′-GGTGACACTATGATGCATATGACGAGGAC-3′ (SEQ ID NO:12)) were used to amplify the RCA product by PCR and to restore the T7 promoter sequence at the Lassos 3′-end so that the products could be transcribed in vitro. Since this PCR reaction sometimes yields multiple products, the DNA fragment corresponding to the monomer Lasso sequence was gel-purified. The resulting DNA template was used for transcription of selected Lasso RNAs.
The internally 32P-labeled Lasso RNAs were incubated at 37° C. with TNF2 target (709-nt fragment of TNF mRNA) in buffer containing 50 mM Tris-Cl (pH 7.5)/10 mM MgCl2/20% formamide (standard binding conditions). The resulting complex was isolated by denaturing PAGE. The band corresponding to the Lasso-target complex was localized on the gel by autoradiography, then excised and eluted. The eluted Lasso complexed with the target was then amplified by RT-PCR as described above.
The sequence of an RNA Lasso scaffold is designed to contain a partially randomized hairpin ribozyme domain, a randomized regulatory element to select for target-dependent circularization, and directed antisense sequences. Using the 229-SN Lasso mini-library as a scaffold, an RNA Lasso library comprising partially randomized ribozyme and regulatory sequences, and a directed antisense library is designed. Restriction sites (XhoI and BamHI) flank either side of a 20-nucleotide antisense cassette sequence, which is supplied by the directed library. The Lasso contains a 10 nucleotide randomized region downstream of the BamnHI site and loop, which constitutes the variable allosteric regulatory element that is optimized through iterative rounds of selection and amplification. The 5′ end of the ribozyme core is also partially randomized to allow for proper processing of the 3′ end of the Lasso molecules induced by the binding to the target. Throughout the Lasso, nucleotides essential to hairpin ribozyme activity (see
Based on the structure of the RNA Lasso library, a DNA library cassette encoding ribozyme, regulatory sequences, and restriction sites allowing the insertion of the directed antisense libraries in desirable orientation is designed and synthesized. This DNA library cassette is prepared in two halves to prevent PCR amplification of cassettes without the directed library insert. The first half contains the T7 promoter sequence, the hairpin ribozyme domain, and the XhoI restriction site. The second half includes the BamHI site, the regulatory element and the 3′-end of the Lasso. Each template segment contains an arbitrary sequence adjacent to the restriction sites to enable efficient digestion.
Directed DNA libraries encoding up to 20-nt-long sequences complementary to TNFα, flanked by restriction sites to allow insertion into the DNA library cassette, using the method described in WO 03/100100A and VLassov et al. (2004) Oligonucleotides (in press). Two hemi-random probes consisting of a defined sequence (PCR primer and restriction site sequences) and I 0-nt randomized regions are prepared. The hemi-random probes are hybridized to TNFα cDNA and adjacent probes are ligated with DNA ligase. The ligation products are amplified by PCR, followed by digestion with the appropriate restriction enzymes.
A target cDNA, comprising the sequence of the target mRNA, is prepared using plasmid PGEM-4/TNF, encoding MuTNFα, by asymmetric PCR with an unmodified primer. Alternatively, regular exponential PCR with a biotinylated primer can be used with subsequent ccDNA strands separation on streptavidin magnetic beads Dynabeads M-280 Streptavidin (Dynal, 20001). In-he first step, the dsDNA is immobilized on the beads due to biotin-streptavidin binding. The mixture is then be heated to separate the DNA chains: non-biotinylated strands appear in the flow-through, while the biotinylated strands remain attached to the beads.
Two hemi-random DNA probes are designed and synthesized, comprising sequences of the defined PCR primer (20 nt) and restriction sites, Xho I & Bam HI (6 nt), with a randomized region (10 nt). Also, masking oligonucleotides that are complementary to the constant regions of the probes are prepared. The hemi-random probes (with constant regions protected with masking oligonucleotides) are hybridized to TNF-α cDNA and the adjacent probes are ligated by T4 DNA ligase at 25-40° C. as described by Kazakov et al (2002). The ligated probes are amplified by PCR using specific primers.
The Lasso DNA library cassettes are digested with appropriate restriction enzymes, and ligated with the digested directed antisense library. The ligated products are amplified by PCR and transcribed to prepare an RNA Lasso library.
To combine the directed library with the DNA library cassette halves, two halves of the DNA library cassette and the directed library species are digested with the appropriate restriction enzymes to generate cohesive ends. The digested products are gel-purified and ligated by DNA Ligase. The ligated product is PCR-amplified using primers that are specific to the fill-length ligated product. The amplified DNA molecules are gel-purified and used as templates for transcription of the Lasso RNA library as schematically presented in
An RNA Lasso library as described above is incubated with target, followed by isolation of complexes on an affinity column, selective amplification of circularized Lassos from Lasso-target complexes, and transcription of RNA from the PCR products. After several additional rounds of selection, surviving members of the library are cloned, sequenced, re-synthesized and tested in the binding assays. The RNA Lasso libraries are incubated with the target TNFα mRNA. The resulting complex is isolated using biotinylated ccDNA complementary to TNFα mRNA immobilized on streptavidin-coated magnetic beads as described previously (Deyev et al., 1984; Stiege et al., 1988; Dynal, 2000). To prevent enrichment of unrelated RNAs (false-positive) that can non-specifically bind beads and undergo self-circularization in a target-independent manner, a counter-selection with is performed with blank magnetic beads or non-specific RNA target (e.g., biotinylated IL-1 ccDNA). After a brief incubation, the beads are washed intensively to remove non-bound and non-specifically bound molecules. Then, Lassos that are specifically bound to the target RNA are eluted.
The eluted Lassos complexed with the TNF target are amplified by RT-PCR as described above. If amplification proves difficult with intact Lasso-target complex, the Lasso-target complex may be dissociated prior to primer extension under highly denaturing conditions that retain the integrity of circularized Lasso. After the first round of selection, the Lasso DNA library is transcribed into the Lasso RNA library. Lasso RNAs are desalted by gel filtration (for example, on a G-50 micro-spin column) and incubated again with the target TNF-α mRNA. The Lasso-target complexes are isolated as described above, and the circularized Lasso RNAs are passed through several additional rounds of selection.
After the last round of selection, around 50-100 resulting DNA fragments are cloned and sequenced. The sequences obtained are assigned to the TNF target sequences and statistically analyzed.
This application claims the benefit under 35 U.S.C. §119 of U.S. Provisional Application No. 60/482,653, filed Jun. 25, 2003, which is hereby incorporated by reference in its entirety.
This invention was made in part during work supported by grant no. R43GM068225 from the National Institutes of Health.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US04/20589 | 6/25/2004 | WO | 5/30/2006 |
| Number | Date | Country | |
|---|---|---|---|
| 60482653 | Jun 2003 | US |
