The following is the current status of a utility patent to be filed by year's end. This is a substantial refinement of the report published in Jan 2004 in this journal. Your comments are most welcome.

ABSTRACT OF THE DISCLOSURE

This invention is concerned with target-binding ligand groupings, and combinatorial libraries thereof. Preferably these ligand grouping result from stable duplexes of hybridized ligand-conjugated polynucleotides (oligonucleotides). These species, termed Polygands (derived from helixes of POLYnucleotides conjugated to liGANDs) have considerable and novel utility, including use as bioactive agents particularly drugs, in diagnostics, and in the construction of supramolecules and nanostructures. Also disclosed is a process of obtaining a ligand grouping which binds to and/or modulates the activity of a target.
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CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and incorporates by reference, U.S. Provisional Application Ser. No. 60/532999 filed Dec 30, 2003 and entitled "High Technology Inventions" by James Saba.
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BACKGROUND OF INVENTION

Neri et al teach helixes of multiple ligand-conjugated polynucleotides. They do not teach a polynucleotide antiparallel duplex hairpin whose 5' and 3' termini at one end are conjugated to separate ligands. Finally, the do not teach utility of the helixes as bioactive agents, or in the construction of supramolecules or nanostructures.

Numerous studies teach duplexes of ligand-conjugated nucleic acids, wherein ligands are longitudinally (relative to the axis of the helix) conjugated at opposite ends of a duplex (1-8).

Kruz, et al (9) teach multimers of protein-conjugated RNA. In each and every of their extensive examples the protein is conjugated to the 3' end of the RNA, which is do to the RNA encoding its conjugated protein. The consequence of all the proteins conjugated to 3' termini is that two such conjugates cannot hybridize to each other to form linear antiparallel duplex and a lateral grouping of ligands. For two proteins to be laterally grouped, they require a third associating polynucleotide which hybridizes to both protein-conjugated RNAs (their Drawing Sheets 2 & 3).

Further distinctions between the present invention and that of Kruz, et al include 1) they do not specify using ligands other than proteins; 2) they do not specify encoded or support-affixed combinatorial libraries, particularly those which are arrayed; and 3) they do not teach utility of the RNA as a bioactive agent, nor as a means of constructing supramolecules or nanostructures.

Miculka, et al (10) teach dynamic libraries of ligand-conjugated polynucleotides wherein the polynucleotides only hybridize to form a stable helix when bound to target. Specifically they state in paragraph [0005] "combination of the molecular species (ligand-conjugated polynucleotides) present in the substance library take place only in the presence of the substrate molecule (target)". Preferably (their Figure 1) such a supramolecular complex consists of three ligand conjugated polynucleotides wherein the ligands cradle the target at the center of the helix. Their polynucleotide conjugates cannot be support affixed since complexing with target requires their dynamic combination. As with Kruz, et al there is no mention of the utility of the polynucleotides other than in complex formation.

Espanel, et al (11) teach arrays wherein each locus contains two peptide ligands, and their utility to analyze protein-protein interactions. Each array locus contains inhomogeneous groupings of ligands since the distribution of the two peptides within the loci is random.

Skaliter et al, Lam et al, Liu et al, Golebiowski et al, and Nestler, et al (12-16 teach ligand libraries and their use in studying molecular recognition, in finding viable drug candidates, cell surface receptor ligands, nuclear receptor ligands, enzyme activity inhibitors and substrates, MHC anchor residues, lymphocyte and antibody epitopes, and peptide vaccines.
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BRIEF DESCRIPTION OF DRAWINGS

Figures 1A-1D. Polygand examples.
Figure 2. Polygand bound to a target via two ligand-binding sites.
Figure 3. Polygand whose two identical ligands bind to two identical receptors on two targets.
Figure 4. Polygand functioning as a decoy.
Figure 5. Supramolecule of polygands.
Figure 6. Formation of soluble nondynamic combinatorial polygand libraries.
Figure 7. Formation of arrayed polygands.
Figure 8. Formation of arrayed and crosslinked (hairpin) polygands.
Figure 9. Array of ligand groupings, formed via association modules.

DETAILED DESCRIPTION OF INVENTION

An entity is "distinct" when in some intrinsic characteristic it is different from others. For clarity, when describing a process involving a mixture of distinct molecules, only one of each distinct molecule is being referred to. An unqualified statement such as "molecules" optionally indicates a multitude which are identical or distinct.

A "molecule" is the smallest particle of a substance that retains all the properties of the substance, and is composed of an exact number, kind and arrangement of atoms. The atoms thereof of often but not necessarily covalently associated.

A "molecular segment" is a portion of a molecule.

A "bioactive" molecule or molecular segment alters biological functioning in a relatively specific fashion. Such species of course include drugs, which are used or anticipated to be useful in the treatment or prevention of disease in animals and plants.

It is convenient to speak of generic groups of molecules and molecular segments. Major generic groups include nonpolymeric organics, polynucleotides, nucleotides, polypeptides, and polysaccharides.

"Nonpolymeric organics" include a vast number both natural and synthetic molecules and molecular segments.

A "polynucleotide" is a single-stranded polymer which comprises nucleotide monomer side-groups which can match up (hybridize) with those of a complementary polynucleotide to form a linear helix (duplex, triplex, or quadruplex). In addition to the naturally occurring nucleotide monomers, there are a vast number of known and conceivable derivatizations, and polymerized combinations thereof. Relatively short polynucleotides are often called oligonucleotides or oligos. A "nucleic acid" consists of one or more polynucleotides.

Polynucleotides are particularly valuable because of their inherent encoding of readily accessible information. Furthermore, polynucleotides can be designed to be relatively featureless, unreactive chemically and immunologically, and of variable hydrophobicity. Thus making them compatible with various chemical processes and therapeutic protocols. Lastly, complementary polynucleotides hybridize to form rigid duplexes making them excellent ligand scaffolds.

A "polypeptide" is a polymer whose monomer linkages are usually but not necessarily peptide bonds, and whose side groups are usually but not necessarily selected from the naturally occurring amino acid side groups such as hydrogen (glycine), methyl (alanine), etc.. Numerous unnatural polypeptide derivations, with modifications to both the amine linkages and side groups are known. Relatively small polypeptides are often termed peptides.

A "protein" is a naturally-occurring polypeptide of any length.

Although the distinction between polynucleotides and polypeptides is becoming less clear with time, for the purposes herein, polypeptides lack complementarity of their side groups and consequent inability to form linear helical duplexes.

"Ligand-conjugated association modules" or "association modules" complex such that their conjugated ligands are grouped. Association modules do not comprise protein, and are preferably polynucleotides.

A "ligand grouping" is a stable noncovalent association of multiple ligands, wherein this stable association is the consequence of, and is dependent upon, the complexation of ligand-conjugated association modules.

Ligand grouping bind, or are prospective to bind, one or more targets in a relatively specific fashion. A ligand usually contacts a target at one location, yet certain ligands can contact a target at multiple sites, or even contact multiple targets. While binding is usually of a noncovalent nature, covalent bonding can occur.

A ligand is at least 250 Daltons MW, and preferably comprises a polypeptide, polysaccharide, and/or nonpolymeric organic. Often a ligand is not a protein.

A ligand does not comprise a polynucleotide, nucleotide (including derivatives of less than 600 Daltons MW) or a fragment thereof (such as those occurring in the degradation or synthesis of polynucleotides).

Furthermore, signal emitting labels, and chemical synthetic reagents used in DNA-templated reactions are not target-directed ligands.

A ligand may be conjugated to an association module via a linking molecular segment, for example to space the ligand from the association module, or allow selective cleavage.

While a "target" can consist of a variety of substances, preferably targets are proteins, nucleoproteins, polysaccharides, and nonproteasous membrane components; and most preferably naturally-occurring in viruses, cells or organisms; especially those which reside in humans or their infectious agents.

"Targets" do not include nucleic acids which are uncomplexed with protein.

Of essence to the present invention is the "polygand" (duplex of POLYnucleotides conjugated to LiGANDs) which is comprised of a multitude of ligand-conjugated polynucleotides stably hybridized to each other to form a helix wherein the ligands are laterally grouped, and often proximal.

Preferably ligands are conjugated to the 3' and 5' terminal nucleotides at one end of the antiparallel duplex.

It is understood that the duplex of a polygand need not consist of perfectly complementary polynucleotides, and that mismatches and bulges may occurs to a limited extent.

Examples of polygands are sketched out in Figure 1. The ligands represented by dark lines in these and many subsequent figures are polymers, preferably polypeptides. Note the ligands are "laterally grouped" relative to the helix axis and that their spacing is always less than the length of the duplex axis. Ligands are "proximal" if they can readily noncovalently contact or approach each other at less than 25 Angstroms when in solution and not bound to target(s).

In the polygand of Figure 1A the two laterally grouped polymeric ligands are conjugated to the 3' and 5' ends of an antiparallel duplex, and are in contact. Note that the 3' and 5' ends of the duplex need not be flush.

Also seen in Figure 1A is the highly useful optional polynucleotide tail. One utility thereof is as an code, allowing detection, identification, and sorting of polygands. For example, such encoded polygands could be detected and identified by a primer-based amplification such as PCR or Rolling Circle Amplification, or addressed to an array. This polynucleotide tail can also be bioactive.

Note the complexes described by Kruz, et al could not be utilized in primer-based amplifications since it is the 3' end which is preferably conjugated to the protein.

In the polygand of Figure 1B the two grouped ligands are nonpolymeric organic molecules.

In the polygand of Figure 1C the polynucleotides have been covalently crosslinked by a polynucleotide loop at the duplex terminus distal the ligands, resulting in a hairpin. Crosslinking the polynucleotides usually imparts stability and can be achieved by other means including a psoralen, a platinum complex, a reactive polynucleotide derivative, or a chemical linker. Also note in Figure 1C one polynucleotide terminus is conjugated to two ligands, one being cyclic.

Figure 1D exemplifies a polygand wherein the ligand grouping is not at a terminus.

As seen in the triplex Figure 1E, the helix need not be duplex. Alternatively to this example, only two polynucleotides of this triplex need be conjugated to ligands.

Note the similarity of certain polygands to antibodies or antibody binding sites, and the truly vast number of combinations possible. Recognize further that almost all antibody-based technologies can be analogously applied to polygands.

As with antibodies any of a large variety of other molecular segments can be affixed to polygands, particularly at the helix terminus distal the ligands. Included therein are labels and therapeutics including prodrug-activating enzymes, toxins and radioisotopes.

Figures 2-4 show sketched examples of polygands interacting with targets.

In Figure 2 one polygand binds at two loci in one target. Note the capacity of the duplex terminus to dynamically break and reform base-pairs ("breath"), and that this can be modulated.

An important objective of current drug research is the advantageous linking a known drug with various molecular segments attempting to increase a drug's efficacy. As seen in Figure 2, polygands are well suited to finding such combinations, wherein at least one the ligands may be known to independently bind a target.

Another objective of current drug research is dimerization of target-binders so as to modulate the association of targets. Polygands also are well suited to achieve this as shown in Figure 3.

Once the complexes as in Figures 2 & 3 are characterized, a relatively small ligand linking group could be designed such that polynucleotides are no longer required, perhaps imparting greater bioavailability.

Polygand bioactivity may also reside in the polynucleotides, particularly as a "small bioactive nucleic acid" which contain less than 500 total nucleotides, and preferably less than 200 total nucleotides. The most important small bioactive nucleic acids are 1) antisense oligonucleotides; RNase L activating 2-5A antisense chimerics; triplex-forming); chimeric RNA-DNA oligonucleotides; small interfering RNA (siRNA); siDNA; siDNA:RNA hybrids; small hairpin RNA (shRNA); micro-RNAs (miRNAs); small temporal RNAs (stRNAs); decoys; catalytic polynucleotides such as ribozymes; and immune stimulating CpG oligos.

Of paramount importance to the present invention is to identify conjugated ligands which facilitate functioning of these exceptionally promising agents. Conveniently, effects of conjugated ligands on the functioning of some of these agents has been reported.

Figure 4 exemplifies one of the many ways how such facilitation could occur, wherein the polygand ligand grouping facilitates the binding of a decoy small bioactive nucleic acid, perhaps to a transcription factor.

Of course targeting a polygand to a cell or infectious agent and aiding in penetration thereof, are other means of facilitating a small bioactive nucleic acid.

Polygands can be complexed, forming supramolecules and nanostructures. In addition to being investigated for utility in biology, physical scientists are intensely investigating these complexes for the purpose of fabricating miniature electric circuitry and machines.

Figure 5 exemplifies a supramolecular complex wherein three identical polygands have been hybridized to a branched polynucleotide template via encoded tails. Note the similarity of this complex to multimeric immunoglobulins IgA and IgM.

There are numerous variations of forming such supramolecules, and nanostructures of a multitude of such structures. For example, complexed polygands, and the polynucleotide sequences of the branching template, need not be identical. Further, the branching template may have any number of branches, any number of which may be ligand targets. Lastly, polygands may directly target each other.

Of paramount importance to the present invention are static combinatorial libraries of polygands, each member having a distinct ligand combination and/or distinct polynucleotides.

Figure 6 sketches the formation of such a polygand library, each member having a distinct ligand group. We start with a multitude of identical polynucleotides and by various means create a set of ligand-conjugated polynucleotides each having a distinct ligand. In a separate reaction at the right, a multitude of identical polynucleotides, complementary to the first, are conjugated to identical ligands. A library of distinct polygands is formed simply by hybridizing the complementary ligand-conjugated polynucleotides.

Note that the first polynucleotides could have distinct encoded tails, which as mentioned previously have considerable utility. Note also that distinct ligands could be conjugated to both complementary polynucleotides, such that the polygands would not have a common ligand.

An interesting alternative to using two complementary polynucleotides as in Figure 6, is to use one hairpin polynucleotide, both ends of which being ligated using distinct chemistry. The final product is considered a polygand whose duplex terminus distal the ligands is crosslinked by a polynucleotide loop.

Conjugation of ligands to polynucleotides is an incredibly active research. Of course one method of forming such a conjugate is to directly conjugate a preformed ligand and polynucleotide. Another interesting means of conjugation involves using conjugated adenosines as transcription initiators.

A ligand may comprise a linker, perhaps as a means of separating the target binding portion from the polynucleotide, or as a means of cleaving the ligand-conjugated polynucleotide.

Synthesizing large numbers of distinct ligand-conjugated polynucleotides can readily be accomplished. For example, a ligand-conjugated polynucleotide could be completely synthesized by step-wise addition of monomers, or one portion of a ligand-conjugated polynucleotide could be used as a starting point for the stepwise synthesis of the other.

By simultaneously elongating the ligand and polynucleotide, one can tag each distinct ligand with an encoded polynucleotide tail.

Often it is advantageous to affix a polygand to a support such as a bead (microsphere), fiber, or biochip. Particularly it is valuable to have an array (often termed microarray) of distinct polygands. Figures 7A, 7B, 8A & 8B exemplify some of the many possible methods of constructing such polygand arrays.

In Figure 7A, three identical polynucleotides with distinct conjugated ligands are each affixed at one of three loci. While only one probe is shown at each array loci, it is understood that several probes are present at each loci, and that they can be spaced appropriately so that identical probes within one loci are not proximal. While the polynucleotides are shown to be directly affixed to the support, they may be attached to the support via a linker or spacer.

This array is then contacted with a second set of identical and complementary ligand-conjugated polynucleotides under hybridization conditions to form the polygands.

Note the significant potential for diversity in that the ligand of the second set of identical ligand-conjugated polynucleotides can be changed. For example assume we have fabricated 1000 identical biochips, each arrayed with 50,000 polynucleotides conjugated to a distinct ligand. Buy hybridizing each biochip with a different second set of complementary and identical ligand-conjugated polynucleotides, we readily create 50,000,000 ligand combinations.

Stabilization of such hybrids could as previously mentioned could be achieve by a cross-linking the hybridized polynucleotides.

In Figure 7B the distinct polygands are first formed in solution as in Figure 2, and then each is addressed to a array loci via hybridization of an encoded tail with a complementary arrayed probe. Contacting the polygands with a target, for example a cellular protein, could be effected prior to or after addressing.

In Figure 8A, a small stem-loop at the base of the initial affixed ligand-conjugated polynucleotides allows for ligation (enzymatic or chemical) of hybridized complementary ligand-conjugated polynucleotides.

In Figure 8B one end of each of three identical support-affixed duplexes, crosslinked at one end by a loop of polynucleotides, is conjugated to distinct ligands. Subsequently the remaining free termini are conjugated to a common ligand.

Arrays of polygands are novel in contrast to those of Espanel, et al in that the groupings of ligands at a particular locus are homogeneous. Note one can space individual ligand groupings at any desired distinct within an array loci.

Combinatorial polygand libraries can be screened for target-binding and bioactivity. Some particularly important screening applications include, cell receptor activation assays, cell adhesion assays, cell or subcellular entry assays, protein-protein interaction assays, protein modification assays such as phosphorylation, identification of antibody and T cell receptor epitopes and mimotopes, vaccine production, and to find ligands which facilitate the functioning of small bioactive nucleic acids.

Numerous screening schemes using both soluble and support-affixed polygands are conceivable. For example one could simply contact an array of distinct polygands with a target, say a cellular protein receptor, and then determine the loci to which the target bound (perhaps by using a labeled target, a labeled monoclonal antibody to target, or surface plasmon resonance).

Another method of screening involves contacting a target (perhaps present on a bead, in a cell or a particular organ) with a soluble polygand mixture, each distinct polygand having an encoded tail. Subsequent to washing unbound polygands, those bound could be identified via their encoded tails.

A novel means of screening small bioactive nucleic acid-containing polygands depends on the capacity of these polygands to trigger the cellular expression or repression of a label such as green fluorescent protein. Individual assays for each polygand library member would identify ligands which facilitated functioning of the label-triggering small bioactive nucleic acid.

Once identified these facilitating ligands could be transferred to other bioactive agents, particularly to other small bioactive nucleic acids. Alternatively such a facilitating ligands could be conjugated in an identical manner to create and active library of polygands with distinct polynucleotide duplexes.

Of interest in performing a large number of individual assays is a process in which two arrays are brought into close apposition such that reactants on the surfaces of two arrays come into contact, optionally involving liberation of support-affixed reagents.

Worthy of mention is work utilizing fluorescence in the study of whole life animals. By utilizing a fluorescent label-triggering small bioactive nucleic acid, once could examine the capacity of conjugated ligands to target this label-triggering small bioactive nucleic acid to a particular tissue or organ.

Polygands are a particular and preferred class of a broader class of complexes of the present invention. A member of this broader class of complexes can be defined as a stable complex of multiple ligand-conjugated association modules, wherein the ligands are proximal and at least of them is not a protein. The polynucleotides of polygands are just one type of association module. As with polygands, these complexes can be support affixed, perhaps arrayed as sketched in Figure 9.

These examples and accompanying figures have deliberately been made exceptionally simple so as to clearly and concisely present the invention. Further information can be found in the accompanying Provision Patent Application No. 60/532999 filed Dec 30 2003 entitled "High Technology Inventions" by James Saba.

Many modifications and variations of the present invention are possible, and it is intended that all such modifications and variations be included within the scope of present invention as defined by the claims.

CLAIMS

1) A polygand comprising one multiple ligand-conjugated polynucleotide hybridized to itself to form an antiparallel duplex hairpin and a lateral grouping of ligands.

2) The polygand as described in claim 1 wherein the 5' and 3' termini one end are each conjugated to a separate ligand.

3) The polygand described as described in claim 1, wherein one of the ligands is known to bind a target.

4) A library of polygands as described in claim 1, each member having a distinct set of ligands and/or polynucleotides.

5) The library of polygands as described in claim 4, each member being affixed to a separate bead or array locus.

6) A process of obtaining one or more noncovalent ligand grouping which binds to and/or modulates the activity of a target comprising, contacting a library of distinct polygands as described in claim 1 with the target, and subsequently determining those polygands which bind to and/or modulate the activity of the target.

7) A small bioactive nucleic acid, multiple positions therein are each conjugated with a separate ligand.

8) The ligand-conjugated small bioactive nucleic acid described in claim 7, wherein the ligands are distinct and proximal.

9) The ligand-conjugated small bioactive nucleic acid as described in claim 7, further defined as comprising either

i) two ligand-conjugated polynucleotides stably hybridized to each other to form an antiparallel duplex wherein said ligands are laterally grouped, or
ii) one multiple ligand-conjugated polynucleotide hybridized to itself to form an antiparallel duplex hairpin and a lateral grouping of ligands.

10) The ligand-conjugated small bioactive nucleic acid described in claim 7, wherein at least one of the ligands is a known target binder.

11) A library of ligand-conjugated small bioactive nucleic acid as described in claim 7, each member having a distinct set of ligands and/or polynucleotides.

12) A supramolecule or nanostructure comprising a multitude of either

i) two ligand-conjugated polynucleotides stably hybridized to each other to form an antiparallel duplex wherein said ligands are laterally grouped, or
ii) one multiple ligand-conjugated polynucleotide hybridized to itself to form an antiparallel duplex hairpin and a lateral grouping of ligands.

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