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WO2007019119A2 - Complexes d'arn et leur methode de production, capteurs et methodes analytiques associees - Google Patents

Complexes d'arn et leur methode de production, capteurs et methodes analytiques associees Download PDF

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Publication number
WO2007019119A2
WO2007019119A2 PCT/US2006/029810 US2006029810W WO2007019119A2 WO 2007019119 A2 WO2007019119 A2 WO 2007019119A2 US 2006029810 W US2006029810 W US 2006029810W WO 2007019119 A2 WO2007019119 A2 WO 2007019119A2
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rna
helical domain
binding sites
monomeric units
binding
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PCT/US2006/029810
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WO2007019119A3 (fr
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Neocles Leontis
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Bowling Green State University
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Priority claimed from US11/196,003 external-priority patent/US7553945B2/en
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Publication of WO2007019119A2 publication Critical patent/WO2007019119A2/fr
Publication of WO2007019119A3 publication Critical patent/WO2007019119A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention is in the fields of biochemistry and molecular biology, and relates to analytical methods in those fields.
  • RNA tectonics exploits the modular character of natural RNA molecules — which can be decomposed into a large variety of RNA structural motifs — to build new nanoscopic RNA architectures that self-assemble to form nano-assemblies of desired size and shape. RNA is fully programmable and amenable to design by reverse folding. [00004] It is desirable to be able to develop RNA as a medium for (1 ) exploring principles of supramolecular self-assembly and (2) achieving nano-scale molecular design and construction of complex cooperative assemblies capable of realizing diverse functions and practical applications.
  • RNA constructs with increased stability and those that may be readily and reliably applied in analytical methods and devices by using conformational strategies of bind, isolate and detect target biomolecules.
  • the present invention begins with the manipulation of structural motifs in
  • the supporting 2D (secondary) structure is designed and used in turn to specify a compatible and uniquely folding nucleotide sequence.
  • the present invention includes tectoRNA molecules that cooperatively assemble into closed, oligomeric complexes.
  • the present invention also includes directional and conformation-specific self-assembling RNA complexes of various sizes.
  • the present invention includes closed complexes formed by H-shaped tectoRNA molecules based on 4-way junctions (4WJ) and specific lateral stereospecific binding site interactions, such as loop-receptor interactions described herein. These complexes may be assemblies of varying size, geometry, stoichiometry and cooperativity, and may even form expandable oligomeric complexes.
  • the invention includes an RNA complex comprising at least three monomeric units of an RNA molecule, each monomeric unit comprising
  • RNA polymer having a first helical domain that comprises a first overlapping portion (referred to as a helical stacking domain) having a first and second binding site (such as, respectively, a receptor site and a loop site), the first helical domain connected to a second helical domain that comprises a second overlapping portion (or helical stacking domain ) having a first and second binding site (such as, respectively, a loop site and a receptor site), wherein the first binding sites are adapted to binding to one another and are not adapted to bind to the second binding sites, and the second binding sites are adapted to binding to one another and are not adapted to bind to the first binding sites; such that the at least three monomeric units are adapted to self-assemble by respective first binding sites interacting with first binding sites of respective adjacent monomeric units, and respective second binding sites interacting with second binding sites of respective adjacent monomeric units, to form pairs of cognate interactions and so as to form the RNA complex in a circular closed complex.
  • the first and second binding sites may also be two loop sites or two receptor sites with respective pairs of receptor or loop sites on the monomeric unit to be bound, and equivalent variations thereof involving so-called loop and receptor interactions.
  • first and second binding sites bind to one another through non-Watson-Crick interaction.
  • the "loops” are typically terminal loops at the end of the helical domains while the “receptors” are loop structures formed internally within the body of the helical stacking domains as shown in the Figures herein where a given loop L n interacts with a receptor R n where n is an integer used for corresponding identification.
  • binding sites that may be used in accordance with the present invention may also include any structure that is capable of forming, directly or indirectly, site-specific RNA - RNA binding interactions (i.e., interactions that resist dissociation). Examples may include hairpin loops, internal loops and junction loops. These interactions may be of any type including non-Watson-Crick interaction, Watson-Crick interaction or other stereospecific non-covalent interactions. Other examples may include the use of chemical moieties (synthetic or native) attached to the RNA monomers that are likewise capable of stereospecific non-covalent interactions.
  • the invention also includes an RNA complex comprising at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having a first receptor site and a second receptor site, the first helical domain connected to a second helical domain having a first loop site and a second loop site; such that the at least three monomeric units are adapted to self- assemble by respective first receptor sites interacting with first loop sites of respective adjacent monomeric units, and respective second receptor sites interacting with second loop sites of respective adjacent monomeric units, to form pairs of cognate interactions and so as to form the RNA complex in a circular closed complex.
  • the first helical domain and the second helical domain are substantially parallel, and wherein the first helical domain and the second helical domain are connected by a covalently bound bridge portion such that the first helical domain and the second helical domain are held in position with respect to one another.
  • the first helical domain and the second helical domain typically are connected by a covalently
  • the positions of the binding site pairs e.g., comprising the first receptor site, the second receptor site, the first loop site and a second loop site are maintained such that the binding site pairs are spatially compatible in only one possible orientation of the adjacent monomeric units, e.g. where the second receptor site and the first loop site align and where the second loop site and the first receptor site align.
  • This may be accomplished by using different numbers of Watson-Crick base pairs between the binding sites on the monomer units (wherein 11 base pairs provides a rotation of 360 degrees), and/or using elements or structures that change this ratio such that the same degree of helicity may be obtained with more or fewer base pairs, such as through the use of a so-called C-loop structures occurring in natural ribosomal RNAs and described in Nucleic Acids Research 2005 33(8):2395-2409 (April 2005) and which is hereby incorporated herein by reference.
  • the distances between the interacting binding sites (e.g., loops and receptors) from the cross-over point are adjusted so that they are optimal for assembly in only one conformation.
  • first receptor site is incompatible with the second loop site, and the second receptor site being incompatible with the second receptor site.
  • the RNA complex of the present invention may be of any size but typically will be a trimer, tetramer, pentamer or hexamer in a circular series.
  • the invention also includes a method of making a self-assembled RNA complex as described herein comprising the steps: (a) placing in solution at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having first and second binding site (such as, respectively, a receptor site and a loop site), the first helical domain connected to a second helical domain having a first and second binding site (such as, respectively, a loop site and a receptor site); and (b) allowing the at least three monomeric units to react such that the at least three monomeric units self-assemble by respective first binding sites interacting with first binding sites of respective adjacent monomeric units, and respective second binding sites interacting with second binding sites of respective adjacent monomeric units, to form pairs of cognate interactions
  • the first and second binding sites may also be two loop sites or two receptor sites with respective pairs of receptor or loop sites on the monomeric unit to be bound.
  • RNA complexes of the present invention have stable conformations and readily determined molecular weight and size, and accordingly may be useful as electrophoresis markers, and as electron microscopy markers.
  • the present invention also includes an RNA molecule as described, having a first helical domain having a first binding site and a second binding site, the first helical domain connected to a second helical domain having a first binding site
  • RNA Complex Per Se with at least one of the Monomers having an Aptamer [000024]
  • the present invention also includes directional and conformation-specific self-assembling RNA complexes of various sizes that include aptamers, and their application as platforms for improved aptamer-based biosensors.
  • the invention further includes an RNA complex as described otherwise herein comprising at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having a first receptor site and a second receptor site, the first helical domain connected to a second helical domain having a first loop site and a second loop site; such that the at least three monomeric units are adapted to self-assemble by respective first receptor sites interacting with first loop sites of respective adjacent monomeric units, and respective second receptor sites interacting with second loop sites of respective adjacent monomeric units, to form pairs of cognate interactions and so as to form the RNA complex in a circular closed complex, at least one of the monomeric units comprising an aptamer.
  • aptamer refers to reagents generated in a selection from a combinatorial library (typically in vitro) wherein a target molecule, generally although not exclusively a small molecule such as a metabolite or a drug,
  • ⁇ H0707754.1 ⁇ -7 or a protein or nucleic acid is used to select from a combinatorial pool of molecules, generally although not exclusively oligonucleotides, those that are capable of binding to the target molecule.
  • the selected reagents can be identified as primary aptamers.
  • the term "aptamer” includes not only the primary aptamer in its original form, but also secondary aptamers derived from (i.e., created by minimizing and/or modifying) the primary aptamer. Aptamers, therefore, must behave as ligands, binding to their target molecule. Aptamers that bind small molecules have been shown to undergo conformational changes upon interactions with their cognate ligands.
  • a reporter fluorophore introduced into an aptamer in a region known to undergo conformational change can lead to a change in fluorescence intensity or polarization after the binding event.
  • the use of aptamers in RNA constructs is described for instance in U.S. Patents Nos. 6,458,559 and 6,706,481 which are hereby incorporated herein by reference.
  • the aptamer may also select for other types of target molecules, such as organic molecules such as pharmaceuticals, carbohydrate or other biomolecules.
  • the role of the aptamer generally is to allow for a change in the conformation of the aptamer containing RNA of a helical stacking domain such that its binding sites become stereochemical ⁇ better disposed to binding interaction.
  • the first helical domain and the second helical domain typically are substantially parallel, and are connected by a covalently bound bridge portion (i.e., by being connected covalently to form a four-way junction) such that the first helical domain and the second helical domain are held in position with respect to one another.
  • a covalently bound bridge portion i.e., by being connected covalently to form a four-way junction
  • the positions of the respective , first binding sites and second binding sites are maintained such that the first binding
  • the first binding sites may also be constructed so as to be incapable of binding with the second binding sites on neighboring molecules in the complex, to best assure the desired orientation of the adjacent monomeric units.
  • the monomers may be designed so as to self-assemble into trimers, tetramers, pentamers, hexamers, heptamers or octamers, or even more complex closed shapes as described herein.
  • the RNA complex may comprise at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, wherein the first binding sites are adapted to binding to one another and are not adapted to bind to the second binding sites, and the second binding sites are adapted to binding to one another and are not adapted to bind to the first binding sites; such that the at least three monomeric units are adapted to self-assemble by respective first binding sites interacting with first binding sites of respective adjacent monomeric units, and respective second binding sites interacting with second binding sites of respective adjacent monomeric units, to form pairs of cognate interactions and so as to form the RNA complex in a circular closed complex, at least one of the monomeric units comprising an aptamer.
  • the present invention also includes a method of making a self-assembled RNA complex comprising the steps: (a) placing in solution at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having a first binding site and a second binding site, the first helical domain connected to a second helical domain having a first binding site and a second binding site, at least one of the monomeric units comprising an aptamer and a substance that interacts with the aptamer; and allowing the at least three monomeric units to react such that the three or more monomeric units self-assemble by respective first binding sites interacting with first binding sites of respective adjacent monomeric units, and respective second binding sites interacting with second binding sites of respective adjacent monomeric units, to form pairs of cognate interactions and so as to form the RNA complex in a circular closed complex.
  • first and second helical domains are substantially parallel, and wherein the first helical domain and the second helical domain are connected by a covalently bound bridge portion such that the first helical domain and the second helical domain are held in position with respect to one another; and the first binding sites and a second binding sites are maintained such that the first binding and the second binding site are spatially compatible in only one possible orientation of the adjacent monomeric units.
  • the present invention also included biosensors and analytical methods based on cooperative tectoRNA complexes, which may be based upon the effects of RNA self-assembly on the affinity and cooperativity of analyte binding.
  • the present invention also includes a method of determining the presence or absence of an analyte through the formation a self-assembled RNA complex, the method comprising the steps: (a) placing in solution at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, at least one of the monomeric units comprising an aptamer adapted to react with the analyte; and (b) allowing the one or more of the monomeric units comprising an aptamer to react with the analyte, and such that the at least three monomeric units self-assemble by respective first binding sites interacting with first binding sites of respective adjacent
  • the presence or absence of the complex may be determined by any method, such as by a method selected from the group consisting of electrophoresis, chromatography and surface plasmon resonance.
  • the present invention also includes a sensor adapted to bind an analyte, the sensor in general terms comprising: (a) a vessel containing a solution of at least three monomeric units of an RNA molecule, each monomeric unit comprising RNA
  • ⁇ H0707754 1 ⁇ 11 having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, at least one of the monomeric units comprising an aptamer adapted to react with the analyte such that the at least three monomeric units are adapted to self-assemble in the presence of the analyte by respective first binding sites interacting with first binding sites of respective adjacent monomeric units, and respective second binding sites interacting with second binding sites of respective adjacent monomeric units, to form pairs of cognate interactions and so as to form an RNA complex in a circular closed complex; and means to detect the presence or absence of the complex.
  • the detection means may be any means adapted to detect the presence of the closed complex, such as an instrument for carrying out an analytical method selected from the group consisting of electrophoresis, chromatography and surface plasmon resonance, such as those known in the art. This detection may also be facilitated by using fluorescent markers on one of the RNA monomers, as described herein to render the monomers or the complexes they form detectable by fluorescence-based techniques and instrumentation.
  • Another aspect of the present invention is a method of determining the presence of at least two analytes in solution through the formation of respective self- assembled RNA complexes, the method comprising the steps: (a) placing in contact with the solution the surface of two silicon cantilevers bearing respective monomeric units of an RNA molecule of a respective first and second type, each monomeric unit
  • RNA having a first helical domain having a first and second binding site comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, and the monomeric unit of the first type comprising an aptamer adapted to react with the first of the two analytes, and the monomeric unit of the second type comprising an aptamer adapted to react with the second of the two analytes;
  • the present invention also comprises an apparatus for determining the presence of a plurality of analytes in solution through the formation respective self- assembled RNA complexes, the apparatus comprising a plurality of silicon cantilevers, each having attached to its surface respective monomeric units of an RNA molecule, each monomeric unit comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, and each monomeric unit comprising an aptamer adapted to react with a respective one of the plurality of analytes.
  • This apparatus may additionally comprise means for measuring the change in the surface characteristics of silicon cantilevers in response to the formation of an RNA complex in a circular closed complex thereupon.
  • An example is a laser adapted to detect changes in the angle of the surface of the silicon cantilevers by detection of the reflected light from the cantilever.
  • RNA Monomer Comprising Fluorescent Marker and Complexes [000041] Another variation of the present invention is an RNA molecule comprising RNA having a first helical domain having a first binding site and a second binding site, the first helical domain connected to a second helical domain having a first binding site and a second binding site, at least one of the helical domains comprising a fluorescent reporter molecule moiety.
  • These monomers include an RNA molecule as described, having a first helical domain having a first binding site and a second binding site, the first helical domain connected to a second helical domain having a first binding site and a second binding site; such that the at least three monomeric units are adapted to self-assemble by respective first binding sites interacting with
  • RNA complex in a circular closed complex, at least one of the helical domains comprising a fluorescent moiety.
  • fluorescent moiety examples may include fluorescein and tetramethylrhodamine (TAMRA), or various non-fluorescent quenchers.
  • the invention further includes an RNA complex as described otherwise herein comprising at least three monomeric units of RNA at least one of which has a helical domain comprising a fluorescent moiety.
  • RNA complexes of the present invention may be used as analytical markers, such as in analytical schemes described herein.
  • Still another aspect of the present invention includes methods and compositions related to the in vitro selection methods by which RNA monomers can be found that interact with RNA monomers with newly developed binding sites (i.e., loop or receptor sites of a previously unapplied nucleotide sequence).
  • a new binding site sequence desired to be matched is put in place of an already characterized binding site sequence in a first RNA molecule.
  • the corresponding receptor region of another RNA molecule is then randomized to create a library of RNA molecules from which to select molecules that bind to the new site.
  • Positive selection is then carried out by mixing the combinatorial library with the new site-bearing RNA molecule.
  • Complexes such as trimers formed may then be separated by electrophoresis gels, while non-binding RNA candidates are eliminated. The complexes may then be selectively taken from the gel and amplified
  • the invention also includes a method of identifying an RNA molecule capable of forming a self-assembled RNA complexes with RNA molecules of two or more known types, the method comprising the steps: (a) placing in solution: (i) monomeric units of an RNA molecule of a respective a first and second type, each monomeric unit comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, such that the first and second types are adapted to bind to one another and; (ii) a plurality of monomeric units of an RNA molecule of a third type, each monomeric unit comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, the first and second binding sites of the third type first helical domain adapted to bind to respective first and second binding sites of the first
  • RNA complex ⁇ H0707754.1 ⁇ -j Q of the second type second helical domain, and the second binding site of the third type second helical domain being randomized, such that only some of the monomeric units of an RNA molecule of the third type self-assemble with the first and second types by respective adjacent monomeric units forming pairs of cognate interactions and so as to form an RNA complex in a circular closed complex; (b) separating from the solution the RNA complex so formed; and dissociating the RNA molecule of the third type from the RNA complex so as to isolate an RNA molecule of the third type.
  • the method may also include the step of amplifying the RNA molecule of the third type isolated from the RNA complex.
  • the method may further include (d) placing in solution the isolated RNA molecules of the third type with monomeric units of an RNA molecule of respective the first type and a fourth type, each monomeric unit comprising RNA having a first helical domain having a first and second binding site, the first helical domain connected to a second helical domain having a first and second binding site, such that the first and fourth types are adapted to bind to one another; (e) allowing the isolated RNA molecules of the third type to react with monomeric units of the first and fourth types, such that only some of the isolated RNA molecules of the third type self-assemble with the first and fourth types by respective adjacent monomeric units forming pairs of cognate interactions and so as to form an RNA complex in a circular closed complex; and (f) separating from the solution the isolated RNA molecules of the third type that do not so self-assemble with the first and fourth types.
  • This method may also include amp
  • the combinatorial library is subjected to a round of negative selection to eliminate molecules that form trimer complexes when the original RNA monomer from which the "new site" RNA monomer is made (i.e., that having a known binding site sequence in place of the new site) is substituted in place of the RNA monomer containing the new binding site sequence (such as when molecule 11 B is substituted for molecule 11 B(NL)).
  • the present invention helps to address one of the core aims of nanoscience, that being to design, build and analyze objects and devices with nanometer scale features. This is many times smaller than the micrometer scale of
  • Supramolecular self-assembly designates "bottom-up" processes in which pre-formed, molecular components, accessible by chemical or enzymatic synthesis, associate by non-covalent interactions that exhibit sufficient binding affinity and specificity to achieve equilibrium polymerization or oligomerization, and thus offers one of the most general strategies for generating nanostructures (Whitesides & Boncheva 2002; Roco 2003).
  • Base pairing in nucleic acids illustrates three features of templated self-assembly: (1) relatively high binding affinity made possible by multiple, non-covalent interactions, (2) high specificity made possible by directional H-bonding, and (3) formation of predictable 3D structure - the double helix - through strand complementarity.
  • linear nucleic acids e.g. double helical DNA or RNA
  • branching motifs such as 3- or 4-way junctions are used to construct complex structures (Seeman 2003).
  • RNA nanoscience While remarkable progress has been achieved in DNA nanoscience, there are good reasons for developing RNA nanoscience.
  • the presence of the 2'- hydroxyl group endows the minor groove of RNA with the capability to form a variety of tertiary interactions, including specific ones between loops and receptors (see below) making possible modes of interaction not available to DNA.
  • Recent crystallographic achievements have made available to molecular engineers a rich treasure trove of RNA structural motifs including novel RNA-RNA interactions.
  • RNA enzymes ribozymes
  • RNA nanoscience draws inspiration from these complex assemblies and, contributes to a better understanding of their structures, functions and dynamics.
  • aptamers as well as catalytic RNA molecules have been obtained by in vitro selection (artificial evolution) methods. Natural aptamers acting as genetic regulators (“riboswitches”) were recently discovered in untranslated regions of
  • RNA molecules that sense and respond to changes in the concentrations of metabolites (Winkler & Breaker 2003). Binding of the target molecule to the recognition element, usually located in the 5'-UTR of the mRNA, results in allosteric changes of the RNA secondary and tertiary structure. The structural changes modulate gene expression by various mechanisms, including transcription termination, translation initiation, or mRNA processing. The existence of a unique two-domain riboswitch that appears to cooperatively bind glycine molecules indicates that RNA molecules can be as versatile as proteins in their responses to changes in the environment.
  • the glycine- activated riboswitch consists of two tandem glycine aptamers that respond cooperatively to glycine concentration with a Hill coefficient of 1.64 (Mandal et al. 2004). Cooperative binding by protein enzymes, receptors and gene control factors provides cells with the means to respond to small changes in ligand concentrations.
  • the present invention allows one to construct RNA molecules tuned to respond to small changes in analyte concentration in a desired concentration range by coupling analyte binding to RNA self-assembly.
  • RNA presents advantages as a medium for supra-molecular design, owing to its well-defined hierarchy of folding. Natural RNA molecules are 3D mosaics that are resolvable into modular motifs.
  • the modular units can be classified geometrically and functionally.
  • A-form double helices function as rigid struts that connect other modular structural elements, including terminal hairpin loops, internal loops ("2-way junctions"), and multi-helix or junction loops, which serve as branch points, thus making complex molecular architectures possible.
  • Motifs can also be classified according to their functions: some serve to mediate RNA-RNA or RNA-protein interactions, others to bind small molecules or substrates, others to organize catalytic sites, and still others to provide points of flexibility (e.g.
  • RNA structure thus facilitates 3D modular design, while the sharp difference in stability between the RNA secondary and tertiary structure makes RNA fully programmable, allowing for design by reverse folding:
  • the molecular engineer arranges the modular motifs in 3D space so that they can mediate self-assembly in the final design.
  • the engineer designs the secondary structure - the pattern of double helical struts and branching motifs that connect and organize the functional motifs in the individual molecular units that will self-assemble to form the supramolecular structure.
  • the engineer designs the sequences to be synthesized. Each sequence is optimized to uniquely fold into its required 2D (secondary) structure (Hofacker 2003).
  • the advantages of using RNA as a medium for nanoscience are:
  • the rapidly growing sequence and structure databases provide a growing repertoire of sequence-specific tertiary interactions and branching motifs from which to draw for tectoRNA design.
  • RNA molecules that bind a host of small molecules and new catalytic RNAs (ribozymes).
  • RNA backbone allows for great diversity of structures and the design of intrinsically flexible molecules that exhibit adaptability upon self- assembly.
  • Figure 1 shows RNA molecules designed to associate non-covalently through specific, non-covalent loop-receptor interactions, in accordance with the prior art.
  • Figure 2 shows an assembly of RNA molecules with one type of loop- receptor interaction, in accordance with the prior art.
  • Figure 3 shows an assembly of RNA molecules in accordance with the prior art, showing isomerization of the four-way junction resulting in alternative assembly modes.
  • Figure 4 shows a directional assembly of RNA molecules in accordance with one embodiment of the present invention, using two loop/receptor interaction motifs.
  • Figure 5 shows a RNA molecule designed to assemble preferentially by loop-receptor interactions from one of the four-way junction conformations, in accordance with one embodiment of the present invention.
  • Figure 6 shows RNA molecules designed for directional binding from only one four-way junction conformation, in accordance with one embodiment of the present invention.
  • Figure 7 shows RNA molecules of a design that assemble with left/right orientational compensation, in accordance with one embodiment of the present invention.
  • Figure 8 shows RNA molecule assembles with both head/tail and left/right compensation, in accordance with one embodiment of the present invention.
  • Figure 9 shows molecules designed by using three specific loop-receptor motifs to determine the stoichiometry of complexes in accordance with one embodiment of the present invention.
  • Figure 10 shows other molecules designed in accordance with other embodiments of the present invention, to demonstrate stoichiometries up to six, including tetramers, pentamers and hexamers.
  • Figure 11 shows the formation of closed complexes by mixing monomer units that assemble head-to-head with monomer units, in accordance with another embodiment of the present invention.
  • Figure 12 shows that identical hexameric complexes can be formed by three different RNA monomers, in accordance with yet another embodiment of the present invention.
  • Figure 13 shows an example of the relationship between molecules in the three different series of RNA monomers, in accordance with still another embodiment of the present invention.
  • Figure 14 shows an example of an RNA hexameric complex in accordance with still another embodiment of the present invention.
  • Figure 15 shows the design for two-stranded tectoRNA module with a
  • Figure 16 shows the coupling of an aptamer module to loop-receptor structure via a short communication module to couple analyte binding to a cooperative tectoRNA self-assembly, in accordance with still another embodiment of the present invention.
  • Figure 17 shows a tectoRNA structure adapted to be used in a FLISA type assay, in accordance with one embodiment ofthe present invention.
  • Figure 18 shows a tectoRNA structure that may be used as a platform for selecting loop-receptors (RR) for new loops, orthogonal to existing loop receptors, in accordance with one embodiment of the present invention.
  • RR loop-receptors
  • Figure 19 shows a tectoRNA structure that may be used as a platform for selecting cooperative aptamers for molecular targets of choice, in accordance with one embodiment of the present invention.
  • RNA Tectonics principles of RNA supramolecular design
  • RNA Tectonics principles of RNA supramolecular design
  • pairs of non-covalent — and thus reversible — RNA loop/receptor interactions that exploit RNA shape complementarity, base stacking interactions, and formation of non-Watson-Crick base pairs to achieve high affinity and specificity have been created (Jaeger & Leontis 2000; Jaeger et al. 2001). These interactions mediate lateral association of helical elements.
  • RNA assembly generally requires the presence of magnesium ions or other multivalent cations (Jaeger & Leontis 2000).
  • RNA molecules were originally designed such that they formed homodimers as shown in Figure 1 (left panel).
  • Figure 1 shows RNA molecules designed to associate non-covalently through specific, non-covalent loop-receptor interactions, in accordance with the prior art, wherein red indicates the GAAA loop and blue the 11-nt receptor specific for GAAA (Gate et al. 1996; Cate et al. 1996). Black, vertical parallel lines indicate Watson-Crick paired anti-parallel double helices. [000077] RNA monomers ("RNA tectons”), designed with properly spaced interacting motifs, associate with surprisingly high binding affinity (K d ⁇ 4 nM) in the
  • RNA molecules Figure 2, left panel
  • 4WJ four-way junction
  • Each molecule can interact with two others, resulting in oligomers or equilibrium (non-covalent) polymer arrays (filaments) that assemble, as shown in Figure 2 (middle and right panels).
  • Figure 2 shows an assembly of RNA molecules with one type of loop- receptor interaction, in accordance with the prior art. Assembly can occur in head-to- head or head-to-tail fashion and is therefore non-directional.
  • Figure 3 shows an assembly of RNA molecules in accordance with first- generation RNA designs, wherein isomerization of the 4WJ resulted in alternative assembly modes.
  • Figure 4 shows a directional assembly using two loop/receptor interaction motifs. The left panel shows a head-to-head assembly while the right panel shows a head-to-tail assembly. Gold indicates GGAA loops and green indicates GGAA receptors.
  • the second source of inhomogeneity was eliminated by manipulating the positions of the interacting motifs relative to the 4WJ cross-over positions so that the motifs are properly spaced for assembly in only one 4WJ conformation (Nasalean et al. In preparation). It was found that loop-receptor motifs are optimally oriented for assembly when separated by 11 base pairs (Jaeger et al. 2001). Thus, the molecule shown in Figure 5 (center panel) assembles preferentially from the 4WJ conformation shown in the left panel, in which the interacting motifs are separated by 11 base pairs.
  • Molecule 16 produces very long linear arrays, while 11 assembles cooperatively to form compact oligomers of definite stoichiometry. These complexes are trimeric as described herein. (At high concentrations, 11 also forms a higher MW species.) The cooperative oligomerization of 11 was inferred from the failure to observe dimeric species, even at sub-nanomolar concentrations and from competition experiments showing that high concentrations of 16 are required to disrupt 11 complexes (Hassan et al. M.S. Thesis, Bowling Green State University). Moreover, complexes of 11 are quite stable and exchange slowly with added radioactively labeled 11.
  • Figure 6 shows a comparison between two different RNA molecules (referred to as Molecule 11 and Molecule 16) designed for directional binding from only one 4WJ conformation.
  • Molecule 11 assembles without orientational compensation and forms closed complexes.
  • Molecule 16 assembles with head-to-tail compensation to form long fibers.
  • the sequences of these molecules differ at only two nucleotides (shown in red), demonstrating the remarkable control of self-assembly of RNA that is possible with the present invention.
  • tecto RNA molecules are asymmetric, chiral objects, when two molecules such as Molecule 11 assemble head-to-head and right-to-left, the translation of the second molecule is accompanied by rotations that are not compensated when successive units are added and a curvature is produced in the assembly.
  • the curvature is not confined to a single plane and helical arrays result, but when assembly is confined to a single plane, closed complexes result.
  • orientationally uncompensated molecules like Molecule 11 generally exhibit sharp, discrete bands on native gels, and appear as globular clusters or "rosettes" by TEM, indicating that such molecules form closed complexes.
  • each monomer added to the assembly is rotated 180° about an axis parallel to the direction of polymerization.
  • each monomer is rotated 180° about an axis perpendicular to the direction of polymerization.
  • the two successive rotations about orthogonal axes lying in the same plane are equivalent to a 180° rotation about an axis perpendicular to the plane of the monomer unit.
  • Figure 7 shows RNA molecules of a design that assemble with left/right orientational compensation.
  • the asterisk (*) indicates the 5'-end of each molecule.
  • the second molecule must rotate 180° about an axis in the plane of the molecule and perpendicular to the axis of propagation. This design showed that equilibrium polymers are also obtained by left/right compensation.
  • FIG. 8 shows Molecule 29 assembles with both head/tail and left/right compensation. It forms closed complexes like uncompensated molecules (e.g. molecule 11). Molecule 29 is an example of one designed in accordance with the
  • Figure 9 shows molecules designed by using three specific loop- receptor motifs to determine the stoichiometry of Molecule 10 complexes. [000098] Other molecules were designed, as shown in Figure 11 , to form stoichiometries up to six. Thus, molecules 11 A, 11 B, 11 C, and 11 D(4) have the appropriate interfaces to combine to form tetramers, 11 B(5), 11 C, 11 D, and 11 E, and 11 F can combine to form pentamers, while 11 A, 11 B, 11 C, 11 D, 11 E, and 11 F can form hexamers.
  • the present invention also includes a method for developing the constructs illustrated schematically in the first two panels of Figure 11 using 2D tiles that assemble either head-to-tail (compensated) to form linear arrays or head-to-head (uncompensated) to form closed complexes.
  • a method for developing the constructs illustrated schematically in the first two panels of Figure 11 using 2D tiles that assemble either head-to-tail (compensated) to form linear arrays or head-to-head (uncompensated) to form closed complexes.
  • This method accordingly provides for the expansion of the present invention to produce larger complexes consisting of more subunits.
  • Figure 11 shows the expansion of
  • Figure 12 demonstrates that identical hexameric complexes (possibly closed) can be formed by RNA molecules 16 + 11 and by 16 + 40.
  • Structures in accordance with the present invention may be studied to test different sequences of compensated and uncompensated interactions to determine which ones form closed complexes, and to determine the stoichiometries of the complexes.
  • new closed complexes in accordance with the present invention may be identified by native gel electrophoresis and characterized by TEM studies to determine their dimensions and shapes. The relative positions of interacting motifs relative to the 4WJ may be varied to affect stoichiometry, cooperativity and stability of closed complexes.
  • FIG. 13 shows an example of relationship between molecules in the 16A-16F and 40A-40F series to molecules in the 11A-11 F series. Shown are 11A (see also Figure 10), 16A, and 4OA.
  • the 11/16/40 series of molecules share the same positioning of the motifs relative to the 4WJ ( Figure 6).
  • the junction is 5 bp from the motifs in the upper half of each molecule and 6 bp from those in the lower half. These distances are crucial for determining the angles at which monomers associate and thus whether closed complexes form and how large they are. These distances may be systematically varied to determine which combinations give closed complexes and to determine the effects of these changes on the stoichiometries of complexes. This may be done by modifying the 11A-F, 16A-F and 40A-F series of molecules.
  • ITC Isothermal Titration Calorimetry
  • DSC Differential Scanning Calorimetry
  • 11A/11B/11C(3) may be carried out on all pairs and triples (all orders of addition) to determine the cooperativity of the complex.
  • 11 C(3) may be added to the complex of 11 A+11 B and the energy parameters obtained will be compared to the pairwise combinations of 11 A+11 B, 11A+11C(3).
  • the cooperativity of the assembly may be measured by ITC.
  • thermodynamic and biophysical measurements may be carried out to elucidate the behavior of these systems (e.g. DSC, CD, etc.) as desired.
  • the kinetics of monomer exchange for 11A/11B/11C and other cooperative complexes may be measured by studying the exchange of added radioactively labeled monomers to preformed unlabeled complexes, and monitoring by gel electrophoresis. The development of the cooperative trimer system is key to the success of these experiments.
  • FRET experiments may be used to measure conformation changes induced by assembly into closed complexes. For instance molecule 11 A with fluorescein (F) and 11C(3) with TAMRA (T) ( Figure 14) and observe FRET from F to T when the molecules are bound to form dimers. Further, molecule 11 B may be added to see whether increases in FRET efficiency occur, as expected when the cooperative complex forms.
  • Figure 14 shows the 11A/11 B/11C(3) system that permits the study of conformational changes upon closed complex formation using FRET by individually labeling molecules with a donor (F) or acceptor (T) fluorescent molecule.
  • the present invention also permits that design and assembly of molecules with rigid junctions and explore the effects of junction rigidity on stoichiometry, cooperativity and stability of closed complexes.
  • 3D modeling indicates that formation of cooperative complexes by 11 requires a conformational change at the 4WJ to bring the two helical domains close to parallel.
  • reducing the conformational entropy of the 4WJ by making it more rigid may increase the stability of the closed complex and improve tectoRNA derived from 11 as platforms for nanotechnology applications.
  • RNA forms A-type helices sequence and structure designs developed for B-type DNA must be modified for RNA, guided by 3D modeling. These modeling studies using the flexible 4WJ from the hairpin ribozyme, indicate that a DAO type double junction should be feasible with RNA, although the optimal design may not be symmetric with regard to the lengths of the helical elements separating the junction motifs ( Figure 15). While this approach requires synthesizing and assembling two RNA strands to create a single monomer unit, it has the advantage of allowing one to create rigid modular units which like their DNA counterparts are suitable for 2D or even 3D tiling of space. Alternative approaches may also be realized with a single strand.
  • Another aspect of the present invention is to use "kissing" hairpins to create the second bridging helix of the double junction.
  • Crystal structures exist for these motifs, including that of the dimerization initiation site (DIS) of HIV (Ennifar et al. 2001).
  • sequences of hairpin-hairpin pseudoknots from the ribosome such as the pseudoknot formed by the hairpin loops located at 418-423 and 2444- 2449 in the H.m. 5OS ribosome can be used.
  • tectoRNA complexes such as 11A/11 B/11C may be developed as general platforms for selecting: (1) new specific loop-receptor interactions (2) more general RNA-RNA interactions, such as internal loop-internal loop; (3) improved aptamers that bind with higher affinity and cooperativity.
  • Cooperatively assembling tectoRNA molecules may in turn be used as scaffoldings to measure weak tertiary interactions in RNA. This may be applied to quantify weak interactions - for example the cooperative system 11 will be used to measure weak binding of GNRA loops to minor grooves - to quantify the specificity of binding GYGA vs. GYAA to minor grooves of different helices. Accurate knowledge of thermodynamic parameters for RNA tertiary interactions is crucial for advancing tectoRNA science.
  • the cooperative tectoRNA complex 11 A/11 B/11 C(3) provides a novel molecular platform adaptable to a wide range of applications.
  • One of the principal features of the present invention is based upon the harnessing of the cooperative self-assembly of these RNA complexes to drive desired molecular recognition events.
  • RNA aptamers generally bind their targets with considerably lower affinity than do antibodies, their protein cousins. Thus, it is highly desirable to
  • ⁇ H0707754.1 ⁇ 4Q find general ways applicable to many different aptamers to increase the affinity of aptamers for their targets. Titration experiments have shown that the trimer formed cooperatively by 11 A, 11 B, and 11 C(3) is more stable than any of the dimers, 11 A/11 B, 11 A/11 C(3), or 11 B/11 C(3).
  • An aptamer module (AM) that recognizes a target of interest can be incorporated into a tectoRNA such as 11 A as shown in Figure 16, positioned close to one of the loop-receptor modules, and coupled by a communication module (CM) (Stojanovic & Koipashchikov 2004).
  • CM communication module
  • Computer modeling and empirical experimentation will determine the best locations for AM and CM - for example flanking a loop receptor as shown or located between a loop receptor and the 4VVJ motif.
  • Figure 16 shows the coupling of Aptamer Module (AM) to loop-receptor R1 via a short Communication Module (CM) in 11A(AM) to couple analyte binding by AM to cooperative tectoRNA self-assembly.
  • the communication module is intended to couple RNA assembly to analyte binding.
  • the analyte binds to and stabilizes the aptamer module by induced fit (Hermann 2000), which in turn stabilizes the loop- receptor module via the communication module (CM), facilitating trimer assembly.
  • MG Malachite Green
  • aptamer modules for non-fluorescent molecules of biological interest such as ATP using a short, unstable helix as communication module
  • Versatile communication modules such as those recently obtained by in vitro selection and shown to couple aptamer binding and enzymatic activity for several different ribozymes (Kertsburg & Soukup 2002), may also be used.
  • the resulting constructs may be studied to determine (1) how RNA self-assembly is modulated by
  • aptamer binding its target (2) the magnitude of changes in aptamer binding affinities; (3) changes in the range of analyte concentration at which the aptamer responds - i.e. its sensitivity to tuning by RNA self-assembly; and (4) cooperativity of analyte binding, especially when aptamer binding sites are also incorporated in 11 B and 11C(3).
  • aptamers such as the malachite green aptamer, which binds and enhances the fluorescence of MG and several of its fluorescent analogues and thus provides for easily detected fluorescent reporting of target binding.
  • FIG. 17 shows a tectoRNA structure adapted to be used in a FLISA type assay, in accordance with one embodiment of the present invention.
  • Figure 17 shows an example of a biosensor one may construct using the present invention, that is analogous to an FLISA-type assay.
  • the first tectoRNA binds the analyte at AM. Another tectoRNA is conjugated to a fluorescent reporter molecule (F). A third tectoRNA is immobilized on the surface of the device. In the presence of analyte assembly occurs and excess fluorescently labeled tectoRNA can be washed off prior to detection.
  • F fluorescent reporter molecule
  • the first and second tectoRNA molecules are free in solution. In the presence of the analyte, they bind to the immobilized tectoRNA; then the well is washed to remove unbound molecules and finally the surface bound fluorescence is measured. This format can be used with many different analytes as the surface bound tectoRNA does not contain the aptamer.
  • the 11A/11 B/11C(3) trimer system provides a platform for systematic in vitro selection and evolution (SELEX) of orthogonal RNA loop-receptor interaction motifs to complement and extend the set of existing motifs already available for tectoRNA. Molecules designed to carry out this selection are shown in Figure 18.
  • the "New Loop" (NL) for which a receptor is to be selected is substituted for L3 in molecule 11 B(NL).
  • the corresponding receptor region of molecule 11 C is randomized to create a library of RNA molecules from which to select molecules that bind to 11 B(NL).
  • Positive selection involves mixing the 11 C(RR) combinatorial library with 11 A and 11 B(NL) in the presence of Mg 2+ ions and running native electrophoresis gels to obtain trimer complexes. Molecules not capable of associating cooperatively with 11 A and 11 B(NL) to form trimers will run faster on the gel and will be eliminated. Both positive and negative selection is possible with this system, allowing one to obtain truly orthogonal loop-receptor pairs. For example, to ensure that the selected receptors do not bind to L3, the combinatorial library 11C(RR) is subjected to a round of negative selection to eliminate molecules that form trimer complexes when 11 B is substituted in place of 11 B(NL). This is done by
  • Figure 18 shows a tectoRNA platform for selecting loop-receptors (RR) for new loops, orthogonal to existing loop receptors.
  • the cooperative 11A/11 B/11C system will also serve as a general platform for discovery of new aptamers for molecular targets of interest, including small molecules, proteins, or nucleic acids of environmental, toxicological, or medical interest. Moreover, it can serve as a platform for optimizing known aptamers to increase affinity and sensitivity in the concentration range of interest.
  • a typical scheme for selection or refinement of an existing aptamer to function optimally within the tectoRNA context is shown in Figure 19.
  • Monomer 11A(RA) in Figure 19 includes module RAM which is coupled to loop receptor R2 by a short communication module.
  • RAM is a randomized region for selecting new aptamers for a target of interest or for optimizing a known aptamer by randomizing at a selected level.
  • the selection procedure may use native gel electrophoresis to select molecules in the library, 11A(RAM), that bind to 11 B and 11C(3) to form trimer cooperative complexes at low (submicromolar) RNA concentrations in the presence of the analyte.
  • Negative selection may be carried out to eliminate molecules in the library that form trimers in the absence of the analyte, or that do not bind the analyte in the desired concentration range.
  • Figure 19 shows a tectoRNA platform for selecting cooperative aptamers for molecular targets of choice.
  • RAM indicates the positions randomized to select for aptamers coupled to the loop-receptor R1.
  • Microfabricated silicon cantilevers coated with gold have been developed recently and used to transduce receptor-ligand binding and DNA hybridization into a direct nanomechanical response - cantilever bending - that can be measured by simple optical detection of the nanomechanical bending of the cantilever (Fritz et al. 2000). Such devices are also described in U.S. Patent No. 6,866,819, which is hereby incorporated herein by reference. [0000135]
  • This nanoactuation mechanism has important advantages, because cantilevers are microfabricated by standard low-cost silicon technology and, by virtue of the size achievable, are extremely sensitive to detect biological interactions.
  • An important advantage of biosensors based on miniaturized silicon cantilevers is that no external probes or labeling is required.
  • Biospecific interactions occur between a receptor immobilized on one side of a cantilever and a ligand in solution. This can cause the cantilever to bend, which is detected optically. Recently, a microarray of cantilevers was reported which was used to detect multiple unlabeled biomolecules
  • tectoRNA for developing effective biosensors using microcantilever technology is therefore clear.
  • expandable tectoRNA complexes have the potential to produce large changes in surface energy and thus sensitively to induce cantilever bending.
  • a tectoRNA monomer that can form either a trimer or a larger (-hexamer) expanded complex can be derivatized with a thiol group on one end to attach it to the gold surface of a microcantilever. It is complexed with unfunctionalized monomer units to form a trimer complex and allowed to react with the surface in the presence of divalent cations, thus coating the surface with trimer complexes.
  • the surface is washed with EDTA solution to dissociate the trimers leaving the monomer unit on the surface.
  • the microcantilever is next treated with a solution containing tectoRNA subunits that can associate with the surface-bound RNA subunit to form expanded complexes (e.g. hexamers). By introducing a specific aptamer module into one of these subunits, assembly can be made to depend on the presence of the analyte of choice. Formation of the
  • the system is modular and can be used with different analytes by using different dissociable aptamer binding tectoRNA. Accordingly, the system may be adapted: (1) To create defined expandable RNA complexes using combinations of compensated and uncompensated interactions, as described above; and (2) To introduce aptamer modules coupled with communication modules to RNA association modules, as described above, to produce tectoRNA that form expanded complexes in response to the presence of an analyte. For this purpose one may use a we 11 -characterized aptamer such as the malachite green aptamer.
  • the present invention may also include modified microcantilever surfaces to create novel, modular, high-throughput, real-time biosensors.
  • RNA may be synthesized by standard techniques using run-off transcription of DNA templates using T7 RNA Polymerase.
  • DNA templates are prepared by PCR amplification of synthetic oligonucleotides which may be designed using a computer program, such as the web application developed for automating the process of converting RNA sequences to DNA template and primer sequences (currently available at http://personal.bgsu.edu/ ⁇ jstomba/index.html).
  • RNA is purified by denaturing gel electrophoresis and radiolabeled with 32P-pCp on the 3'-end or by kinasing on the 5'-end.
  • RNA is routinely purified using preparative denaturing electrophoresis and electroelution followed by alcohol precipitation.
  • tectoRNA constructs of the present inventon may be modeled using computer models assembled manually using software such as Swiss PDB Viewer to manipulate and dock motifs.
  • MATLAB programs may be written to automate the tedious aspects of model building such as the insertion of helical struts, optimal positioning of overlapping helical segments (to correctly position modular motifs) and renumbering of nucleotides at intermediate stages of modeling.
  • the new programs will draw on MATLAB code we have written to identify and extract RNA motifs and non-Watson-Crick basepairs from crystal structures.
  • the computer models may be converted to physical models using rapid prototyping. Additional models for pairs of interacting tectoRNAs, including the 4WJ, for all feasible variations of distances (in basepairs) between the interacting loop and receptor motifs and the 4WJ may be prepared, to assist in the interpretation of experiments. Analytical Methods and Apparatus
  • the self-assembling RNA may be constructed using analyte specific aptamers that allow one to detect the presence of one or more analytes that react with discretion with the incorporated aptamer, allowing the self-assembling RNA only when the analyte(s) combine with monormeric unit(s) having analyte-specific aptamers. This provides a scheme by which one or more analytes may be detected from a common solution if desired.
  • the self-assembly of RNA complexes can be detected using silicon cantilever devices whose surfaces measurably change upon the formation of an RNA complex thereupon.
  • R15-R28 Cate, J. H., A. R. Gooding, E. Podell, K. Zhou, B. L. Golden, C. E. Kundrot, T. R.
  • RNA hydrogen bonds surrounding G+1 and U42 are the major determinants for the tertiary structure stability of the hairpin ribozyme.”

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Abstract

L'invention concerne des complexes d'ARN comprenant au moins trois unités monomères d'une molécule d'ARN. Chaque unité monomère comprend un polymère d'ARN présentant un premier domaine hélicoïdal et un second domaine hélicoïdal présentant respectivement un premier site de liaison et un second site de liaison. Les premiers sites de liaison sont conçus pour se lier à un autre premier site de liaison et ne sont pas conçus pour se lier aux seconds sites de liaison, et les seconds sites de liaison sont conçus pour se lier à un autre second site de liaison et ne sont pas conçus pour se lier aux premiers sites de liaison; de sorte que les trois unités monomères sont conçues pour un auto-assemblage par formation de paires d'interaction endogène, et de sorte à former le complexe d'ARN dans un complexe fermé circulaire. L'invention concerne également des dérivés de ces complexes comprenant des aptamères et des méthodes analytiques, ainsi que des dispositifs faisant appel à ces complexes.
PCT/US2006/029810 2005-08-03 2006-08-01 Complexes d'arn et leur methode de production, capteurs et methodes analytiques associees WO2007019119A2 (fr)

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