WO1996004788A1

WO1996004788A1 - Antisense and triplex therapeutic agents and methods

Info

Publication number: WO1996004788A1
Application number: PCT/US1995/010055
Authority: WO
Inventors: Sidney Altman; Paul Fiedler; Robert A. Levine; Stephen C. Wardlaw
Original assignee: Sidney Altman; Paul Fiedler; Levine Robert A; Wardlaw Stephen C
Priority date: 1994-08-10
Filing date: 1995-08-09
Publication date: 1996-02-22
Also published as: JPH10504819A; MX9700991A; CA2197117A1; EP0774902A1

Abstract

Antisense and anti-gene oligonucleotides and modified oligonucleotides with plural intercalating molecules attached for use in antisense and triplex therapy. Virally infected blood, blood plasma or serum samples are rendered significantly less infectious while maintaining the integrity of the sample for clinical analysis. Synthetic strands of oligonucleotides, and/or nuclease-resistant derivatives of oligonucleotides having intercalating agent molecules attached thereto provide an additive for incorporation into a blood sample which is to undergo analytical tests and procedures. Sterilization of a significant number of intracellular target viruses in the sample renders the sample significantly less capable of infecting those handling it. An appropriate binding material additive, such as an antibody or aptamer, which is specific for epitopes or other binding sites on the free or extracellular target virus, may also be used, either alone or in combination with the intracellular strand ingredients.

Description

ANTISENSE AND TRIPLEX THERAPEUTIC AGENTS AND METHODS

TECHNICAL FIELD This invention relates first to a method and additive for rendering viruses noninfectious in biological samples, such as blood. More particularly, this invention renders viruses contained in in vitro samples of blood incapable of infecting persons handling the samples, and therefore renders the sample significantly less infectious while maintaining the integrity of the sample for clinical analysis. The present invention also relates to the fields of antisense and triplex therapy.

Broadly, antisense therapy involves binding complementary antisense oligonucleotides or modified oligonucleotides to RNA to prevent its translation to disease-related proteins, or to a DNA strand as it is opened by RNA polymerase, thus preventing its transcription to RNA. Triplex therapy involves binding a complementary anti-gene oligonucleotide or modified oligonucleotide to a segment of DNA or RNA double helix to prevent its transcription or reverse transcription. In triplex therapy, the complementary oligonucleotide segment is referred to as "anti-gene," since it may be complementary either to the target strand coding for transcription and translation, or to the complementary or antisense target strand, or possibly to both. BACKGROUND ART

Significant hazards of exposure-induced viral infection, due to the potential infectivity of blood specimens or blood and blood products for infusion (transfusion), exist for health care workers including phlebotomists, laboratory technicians and other health care workers involved in obtaining blood specimens; performing clinical analyses such as routine hematologic analysis such as complete blood counts and others, routine chemical analysis such as determination of enzyme levels, lipid levels, glucose levels, blood urea nitrogen levels and others, routine serologic tests such as test for infectious diseases and blood type determinations, routine immunoassay tests such as the measurement of thyroid stimulating hormone levels and others and routine blood bacteriologic examinations for detecting the presence of bacteria in the blood and other tests on the specimens, or portions thereof or working in cleaning areas where these tasks are performed, or where blood or blood product transfusion containers are handled. Infection may result from exposure to the blood samples, or from plasma or serum which may enter one's body through cuts, abrasions or puncture wounds due to broken or sharp glassware, plastic ware, needles, or other laboratory hardware which is, or has been, in contact with the specimens. Infection may also result from contamination of mucosal membranes or open wounds, either through spills; or by aerosolization of infected samples of blood.

Exposure to contaminated blood may result, for example, in hepatitis infection, or HIV infection, the former being associated with significant morbidity, and sometimes fatality; and the latter being invariably fatal. Bacterial infection of biologic samples is not generally seen as a significant hazard to health care workers, with the exception of mycobacterial infection of sputa.

Procedures are known for inactivating and decreasing the infectivity of biological specimens, which procedures maintain the integrity of the specimen for clinical analysis. Such procedures include photo inactivation of free virus; and exposure of the specimen to ionizing radiation. The former procedure has been performed on human fresh plasma, but this procedure is not applicable to whole blood, and furthermore it requires the active step of exposing the the plasma to light for a period of minutes to an hour, depending upon the volume of the specimen and the intensity of the light. The latter procedure using ionizing radiation is not applicable for use at the point of care where the blood or specimen is drawn or obtained. Other solutions to the aforesaid problems of secondary infection have centered on the reduction of exposure to blood or other liquid specimen aerosols or potentially penetrating objects, generally needles, either by modifying venipuncture techniques; by using latex gloves, masks and goggles by technicians; and/or by using needle guards. Plastic coatings on glass containers or pure plastic containers are often used instead of glass because of a lower likelihood of breakage with the former. The aforementioned attempts to reduce the hazard of blood-borne infection to health care workers are partially effective, but significant hazards remain. Blood products for infusion are screened for hepatitis virus and HIV, but this screening is not 100% effective, and technicians are thus still exposed to virus-positive blood, and blood products. Additionally, technicians who perform the tests on blood samples are, of course, always exposed to the blood.

Aside from the aforesaid solutions, the main foci of dealing with viral infections, having concerned themselves with in vivo prevention by active or passive immunization of exposed personnel, when possible, rather than incapacitating, or significantly reducing, the ability of in vitro sources of the virus to infect others. The quest for a cure, be it a treatment or a vaccine, constitutes the ultimate solution to the HIV/ AIDS problem, as well as other viral infections; however, prevention of further infection of humans exposed to potentially infected in vitro blood or other fluid samples should provide a substantial reduction in the spread of the disease to health care workers. Broad spectrum sterilization of blood or other biological fluid samples by such agents as chlorine, formalin or the like is unsuitable for use with such samples, as these agents irreversibly alter or destroy in a non-specific fashion the very constituents in the sample which are to be measured or infused. The need for a target-specific infection prevention measures has been recognized, but has not been addressed by a widely accepted practical solution.

Prevention of gene transcription or translation can prevent production of disease causing protein, or of protein which represses production of a desirable protein, or of protein necessary for replication of the genome, and hence multiplication of the disease cell, virus, bacteria, protozoa etc. Antisense or triplex binding to an RNA or DNA genome may also directly prevent replication.

The mechanism of action of an antisense oligonucleotide depends on its target oligonucleotide sequence. One mechanism suggested involves binding the antisense oligonucleotide to the point on mRNA where translation is started, thus preventing ribosomes and important initiation factors from binding to mRNA. If protein biosynthesis has already been initiated on the mRNA, the presence of a hybridizing antisense oligonucleotide might block the necessary translocation of the ribosomes along the mRNA.

Antisense oligonucleotides may also inhibit transcription, by binding to a single strand of DNA as it is separated from its complementary strand by RNA polymerase. Inhibition of post-transcriptional process is also possible. In eukaryotic cells, pre-mRNA is subject to a number of maturation processes before mature mRNA is translocated into the cytoplasm for transcription. Antisense oligonucleotides may intervene at any one of these processes, as for example, interfering with intron splicing or 5' end capping. Other translation or transcription mechanisms may be possible, but all share in common the binding of an antisense oligonucleotide to a segment of RNA or single strand DNA. The antisense or anti-gene oligonucleotides may be employed individually, in tandem, or possibly, multiply. Tandem or multiple use of oligonucleotides involves using two or more different oligonucleotides which target for different sequences of DNA or RNA. Use of two oligonucleotides in tandem has been found to yield synergistic results, where there is a relatively short gap between oligonucleotide segments.

The length of the antisense or anti-gene oligonucleotides used may vary dramatically, with some researchers still working with relatively long segments. Most scientists, however, are focusing on relatively short segments. It is currently generally accepted that the oligonucleotides must be at least 15 nucleotides long in order to bind tightly to target sites. Most research has focused on oligonucleotides of 15-50 nucleotides, more often 15-25 nucleotides.

It has been suggested to improve the binding affinity and transport characteristics of oligonucleotides by conjugating them with intercalating agents. Intercalating agents have been attached either to the 3' or 5' end of an oligonucleotide segment to provide additional binding energy, without perturbing the specificity of recognition of the complementary sequence. Intercalating agents provide additional binding affinity in that they are generally planar molecules which are attracted to and insert themselves between adjacent base pairs on double stranded nucleic acids. Various derivatives of acridine are examples of such an intercalating agent. Intercalators that optimize stabilization of double helical segments are generally slightly different from those that optimize stability of triple helices.

SUMMARY OF THE INVENTION This invention relates to additives and methods for preventing specific viruses contained in a mammalian biological sample from infecting persons handling the sample. Suitable additives and methods are described for preventing intracellular and/or extracellular viruses from infecting those handling the samples.

In another aspect, the present invention relates to antisense and anti-gene oligonucleotides and modified oligonucleotides are provided which have plural intercalating molecules attached, in series and/or in parallel. While the terms "series" and "parallel" are not typically used in chemistry or biochemistry, they do provide a convenient description of the structure of the attachment of intercalating agents to oligonucleotide segments in the present invention. In series attachment, a sequence of two or more intercalating molecules joined together are attached to the oligonucleotide segment at the 3' end, and/or the 5' end and/or some place in between. Parallel attachment involves attaching two or more separate intercalating molecules to an oligonucleotide segment at several different points on the segment, e.g., the 3' end, the 5' end, and one or more places in between. A combination of serial and parallel attachment of intercalators may be used.

These and other objects, advantages, and features of the invention will be more fully understood and appreciated by reference to the written specification and appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic illustration of an oligonucleotide sequence having two intercalating agents joined thereto in parallel, hybridized with a segment of DNA;

Fig. 2 is a similar schematic representation, but with intercalating agents joined in series;

Fig. 3 is a representation of an oligonucleotide with a series of two acridine intercalating molecules attached to the 5' end thereof;

Fig. 4 is a representation of an oligonucleotide with a series of two acridine intercalating molecules attached at the 3' end thereof; Fig. 5 is an oligonucleotide segment with a series of two acridine intercalating molecules attached between the ends of the oligonucleotide sequence;

Fig. 6 is an oligonucleotide sequence with two acridine intercalating molecules attached to the sequence in parallel, one at the 3' end and one at the 5' end;

Fig. 7 is an oligonucleotide sequence with parallel acridine intercalating molecules attached at two spaced points between the ends of the sequence;

Fig. 8 is an oligonucleotide sequence having three intercalating acridine moieties attached in parallel, one at each end and one in the middle;

Fig. 9 is an oligonucleotide sequence having three separate series of two acridine molecules each attached in parallel, one at each end and one in the middle; Fig. 10 is an illustration of a triplex formed with an intercalating oligonucleotide sequence;

Fig. 11 is an illustration of an antisense intercalating oligonucleotide hybridized with a single strand of DNA or RNA;

Fig. 12 is a side sectional view of a specimen sampling receptacle into which one or more additives formed in accordance with this invention have been incorporated;

Fig. 13 is a side elevational view of a blood or blood products storage container; and

Fig. 14 is a schematic view of an extracellular viral capsid having a plurality of binding units attached to cell-specific epitopes on the capsid.

DESCRIPTION OF THE PREFERRED EMBODIMENT INTRODUCTION In a potentially infectious blood sample, viruses may exist extracellularly or free or intracellularly as viruses or as proto-viruses such as the HIV virus does. The intracellular viruses or proto-viruses are not susceptible to circulating antibodies because they are protected by the host cells which do not permit penetration of the antibody through the cell's membrane. However, it is possible to inactivate intracellular viruses by the use of the oligonucleotides described herein which can easily penetrate the cell's membrane.

Free viruses have capsids that may or may not be penetrated by oligonucleotides. Such free viruses can be inactivated by preventing their attachment to host cells by the addition of antibody or other suitable binding agent as described hereinafter. This invention provides additives and combinations of additives which can prevent intracellular and/or extracellular or free viruses from infecting persons handling virally infected samples while not destroying or modifying any of the sample's constituents that may be the subject of clinical analysis.

As with all living organisms, viruses include DNA and/or RNA genomes and code for messenger RNA structures which are unique to the particular virus in question. The molecular composition of these genetic structures can be specifically analyzed and the unique nucleotide monomer building block sequences in the single or double helix structures can be determined. In order for a double stranded virus to remain infectious, it must be capable of replication, which involves the formation of new strands which are complementary to the parental "template" strands, thereby producing new double helix genomes. Thus, when the parental double helix in a double helical (either RNA or DNA) genome is replicated, two new double helix genomes will thereafter be formed. In this way, if the double helix is prevented from completely separating into component strands, it cannot replicate, and therefore the virus cannot replicate.

Single strand genomes replicate as follows. A complementary strand is synthesized first by an intracellular mechanism after which a variety of mechanisms may ensue. In all of the ensuing mechanisms, the complementary strand essentially serves as a template for the viral progeny genome strands. If the complementary strand is prevented from either being synthesized or from serving as a template, the single strand genome cannot replicate. If a virus cannot replicate, it will be rendered non- infectious. Additionally, messenger RNA, specific or a given virus, are needed to permit translation of essential proteins. If a virus-unique messenger RNA is enveloped by binding to specific oligonucleotide sequences, the virus is rendered non-infective, if the protein encoded by that messenger RNA is essential for the virus' replication.

In one embodiment of the invention, the additive and method of this invention are operative to render a target virus incapable of replicating by creating localized highly stable, i.e., substantially irreversible under the conditions of use, triple helix complexes on the double helix DNA genomes; and/or by creating localized highly stable double helix complexes on single-stranded RNA or DNA genomes and on messenger RNA encoded by double stranded DNA or RNA genomes or by single stranded RNA genomes. These localized triple and double helix complexes are formed in vitro on intracellular viral genomes, and on RNA, respectively, which are found in infected blood cells, or are found in infected cells in other biological fluids. These localized complexes will begin to form as the blood, blood product, or other biological fluid is drawn into a receptacle containing the additive. The additive which forms the aforesaid complexes, and thus prevents the target virus from replicating, is preferably disposed in the sample receptacle prior to drawing the biological fluid sample, so that the intracellular viral DNA and RNA genomes and messenger RNAs will be disabled relatively shortly after the sample enters the receptacle. Subsequent addition of the additive may be made if so desired.

In one aspect of the preferred embodiment, a multitude of different types of disease can be treated. A DNA or RNA target is identified. An oligonucleotide or modified oligonucleotide which is complementary to the target is synthesized. Two or more intercalating molecules are attached to the oligonucleotide in series or in parallel. The resulting intercalating oligonucleotide is then mixed with suitable pharmaceutical carriers, diluents, and/or other optional ingredients. The resulting pharmaceutical preparation is then administered to the subject to be treated.

THE DNA OR RNA TARGET Virtually any type of disease is susceptible to treatment using the techniques of the present invention. The replication of a genome can be stopped by targeting replication origins in the DNA genome, or in the RNA genome in the case of some viruses. Thus, cancer cells, bacteria, protozoa, and viruses can be kept from multiplying.

Replication can also be stopped indirectly by inhibiting the expression of proteins necessary for replication. The expression of toxins can be inhibited. In some cases, it will be possible to enhance the expression of desirable proteins by suppressing the biosynthesis of a natural repressor or by directly reducing termination of transcription. A DNA or RNA sequence may code for a protein which inhibits production of a desirable protein. Shutting down such a sequence using antisense or triplex therapy is another possible use of this invention. Similarly, one may want to shut down production of a protein which causes or enables causation of a disease.

The inhibition of protein expression can be effected by inhibiting either translation or transcription. Translation can be inhibited by binding an antisense oligonucleotide to the point on messenger RNA where translation is initiated. In the case of eukaryotic mRNA, for example, interference with the interaction between the initiation factor 4F and mRNA is conceivable. A hybridizing oligonucleotide might also block the necessary translocation of ribosomes along the mRNA chain.

Transcription can be inhibited by binding an antisense oligonucleotide to a DNA promoter as the DNA double helix begins to open. Targeting an anti-gene oligonucleotide to the promoter section of a DNA double helix may also prevent RNA polymerase from binding to promoter sequences on the DNA. The oligonucleotide is referred to as anti-gene, in that it may be complementary either to the DNA strand which codes for transcription, or to its complementary strand. In either case, the resulting triple helix will inhibit RNA polymerase binding to the promoter segment.

The inhibition of post-transcriptional processes is also possible. In eukaryotic cells, the primary transcript, the so-called pre-mRNA, is subject to a number of maturation processes before mature mRNA is translocated into the cytoplasm for transcription. In the nucleoplasm, introns are spliced from the pre-RNA and the 5' end of the mRNA is modified to stabilize it. Various bases are altered. The polyadenylation of the mRNA at the 3' end may be linked with the export process. Antisense oligonucleotides may intervene in any one of these processes.

The HIV virus which causes acquired immune deficiency syndrome (AIDS) has target nucleotide sequences in its genome and messenger RNA which can be utilized in the performance of this invention. These sequences include the tat gene; the gag mRNA; the rev mRNA; and the 5' leader sequence, among others.

It is desirable, for purposes of effective and efficient sterilization of viruses, to target sequences in genomes and messenger RNA for specific viruses that are both essential to the reproductive capability of the virus and that are conserved among genetic variations of the virus. Such a target for diverse strains of HIV type 1 is described by Mang Yu, Joshua Ojwang, Osamu Yamada, Arnold Hampel, Jay Rapapport, David Loony, and Flossie Wong-Staal in: "A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1" in Proc. Natl. Acad. Sci. USA.. Vol. 90, pp. 6340-6344, July 1993 as the 5' leader sequence of HIV- 1 since its inhibition would inhibit the expression of both early and late viral gene products due to its presence in all HIV-1 RNA transcripts. OLIGONUCLEOTIDE SYNTHESIS

Once the nucleotide sequence of the target segment of RNA or DNA is identified, the antisense or anti-gene oligonucleotide can be synthesized of complementary nucleotides. Properly sequenced nucleotides that compliment the target nucleotide sequence can be readily produced with a nucleic acid synthesizer of the type sold by Applied Biosystems Inc., for example. Other known oligonucleotide synthesis techniques can be employed.

The length of the oligonucleotide segments should be sufficient to ensure formation of stable triple or double helix complexes at the target DNA or RNA sequences and sufficient to minimize the formation of the complexes at random, non-target locations. Preferably, the targeted DNA or RNA region, and correspondingly the antisense or anti- gene oligonucleotide, has 15-50 nucleotides, more preferably 15-30, and most preferably 20-28. Statistically, the sequence of a 17-mer oligonucleotide occurs just once in the human genome. Thus, an oligonucleotide with 20-28 bases is extremely selective and specific to the desired target segment. The oligonucleotides may be modified to render them more resistant to enzymatic degradation, enhance cellular uptake, or for other purposes. In order to improve uptake by cells and/or resistance to enzymatic degradation, scientists have replaced the negative oxygen on the phosphodiester backbone with methyl or sulfur, creating methylphosphonates or phosphoryl thioates. This will result in an enzyme-resistant synthetic oligonucleotide derivative strand possessing enduring integrity when commingled with a cellular biological material. Nuclease-resistant strands may also be produced by including 2'-O-allyl or 2'-0-methyl groups in the synthetic oligo strands. Phosphoryl dithioates have also been created. Modification by creating phosphate esters and phosphoryl amidates has been accomplished.

Another type of modification which has been investigated is replacement of the phosphodiester bridge between nucleotides with an entirely different group, such as a siloxane bridge, carbonate bridge, carboxymethyl ester bridge, acetamidate bridge, carbamate bridge, and thioether bridge. Besides replacing the phosphate bridge, one can replace the sugar and phosphate residues by a synthetic polymer and thus obtain chemically modified analogues of DNA. The nucleoside units themselves can be modified.

The term "modified oligonucleotide" as used throughout is intended to encompass oligonucleotide segments modified in any of the above suggested ways and in other ways. In order to minimize verbal redundancies in the ensuing description of the invention, the term "oligonucleotide," as applied to the synthesized antisense or anti-gene segment, shall include both oligonucleotides and nuclease-resistant or other modified oligonucleotides. ATTACHMENT OF INTERCALATING MOLECULES It is desirable to create die most persistent triple or double helixes possible so as to render the complexes of antisense or antigene oligonucleotide with DNA or RNA strands, in a practical sense, irreversible. This is accomplished by attaching a plurality of intercalating agent molecules to the oligonucleotide strands, in series, and/or in parallel. An intercalating agent is a generally planar molecule with an affinity for nucleotide bases, such that it tends to insert itself between base pairs along a DNA or RNA strand (Fig. 1). In Fig. 1, for example, the oligonucleotide strand 10 (comprised of thymine nucleotides) includes intercalating agents 11 on the 3' end and 12 on the 5' end, which have inserted themselves between adjacent base pairs A-T and A-T on the complementary DNA strand 20. Exemplary intercalating agents include acridine derivatives, oxazolopyridocarbazole chromophore (OPC), mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes such as LDS-751 available from Exciton Co., anthracycline compounds such as doxorubicin, benzo (e) pyridol indole, furocumarin, daunomycin, 1,10-phenanthroline, phenanthridinium, porphyrin, ellupticine or ellipticinium, derivatives Of the foregoing and/or other related planar aromatic compounds or derivatives thereof. The term "intercalating agent" as used herein is also intended to include groove binders, such as Hoechst-33258 available from Aldrich Co. , Milwaukee, Wisconsin, and thiazole yellow and derivatives thereof. The series addition of intercalating agent to the oligonucleotide involves attaching a chain of two or more intercalating agents, such as an acridine derivative, to one point on the oligonucleotide. Preferably, the intercalating agents in a series chain are separated by a spacer, or are joined to the series chain by some type of spacer 13 (Fig. 2). This establishes sufficient spacing between adjacent intercalating agents that they can insert themselves between every other, or greater, base pair. The nucleotide strands tend to resist insertion of intercalating agents between adjacent base pairs. This is known as the "nearest neighbor exclusion principle." These spacer bridges are preferably aliphatic moieties, most preferably comprising 3 to 5 carbon atoms. In Figs. 3-5, a series of acridine molecules are attached to the 5' end (Fig. 3), the 3' end (Fig. 4), and the middle (Fig. 5) of an oligonucleotide chain, respectively.

The parallel connection of intercalating agents at several points along the length of an oligonucleotide sequence is illustrated in Figs. 6, 7 and 8. In Fig. 6, acridine is connected to both the 3' and 5' ends of the sequence. In Fig. 7, it is attached at two spaced points along the length of the oligonucleotide, but between the ends thereof. In Fig. 8, it is attached to both at the ends and between the ends of the sequence. In Fig. 9, a combination of series and parallel attachment is illustrated. In order to satisfy the "nearest neighbor exclusion principle, it is preferable that any two parallel intercalating moieties be joined to nucleotides which are separated by at least one intervening nucleotide.

The intercalating agent molecules when connected to the synthetic additive strands, as shown above, can provide enhanced binding energy for forming the resultant substantially inseparable double and triple helix complexes. The intercalating agent molecules can be attached to the several locations on the synthetic strands by several procedures for the synthesis of intercalating agent modified oligonucleotides.

Oligonucleotides bearing pendant intercalating acridine derivative molecules may be prepared by use of standard solid-phase synthesis procedures and b-cyanoethyl or methyl phosphoramidite monomers. The intercalating acridine derivative molecules will not react with the reagents employed commonly during solid-phase oligonucleotide synthesis and can therefore be introduced without special "protecting groups. " Reagents that append intercalating acridine derivatives to the 3' end or the 5' end of an oligodeoxy- or an oligoribo- nucleotide are available commercially from Glen Research Corporation, Inc., Sterling, Virginia. These reagents are formulated such that they may be used with most standard oligonucleotide synthesis machines (such as those available from Applied Biosystems, Millipore, or Beckman) and do not require special manipulations or special personnel. Molecules of this general type may be prepared following standard procedures. The intercalating agent molecules will increase the strength of the interactions between the additive oligonucleotides and target viral genomes, and/or target viral messenger RNA, either or both of which may be disposed in the cells, and will cause the formation of complexes which are highly stable and substantially irreversible under the conditions of use of the invention. In this manner, localized enduring triple or double helix complexes will be formed in the genetic material of the virus. These localized triple and double helix complexes will prevent the viral DNA or RNA from replicating, and will prevent viral messenger RNA from being translated into protein. These complexes render the intracellular virus non-infectious so long as the complexes exist. Because there is genetic diversity among various strains of virus it would be preferably to have, as target sequences, more than one target per type of virus so that more effective sterilization may be obtained in cases of genetic diversity among strains of viruses or a mutation in a single target sequence in a genome.

PROCEDURES FOR THE SYNTHESIS OF ACRIDINE MODIFIED OLIGONUCLEOTIDES Oligonucleotides bearing pendant intercalating acridine erivative molecules may be prepared by use of standard solid-phase synthesis procedures and b-cyanoethyl or methyl phosphoramidite monomers. The intercalating acridine derivative molecules will not react with the reagents employed commonly during solid-phase oligonucleotide synthesis and can therefore be introduced without special "protecting groups. " Reagents that append intercalating acridine derivatives to the 3' end or the 5' end of an oligodeoxy- or an oligoribo-nucleotide are available commercially from Glen Research Corporation, Inc., Sterling, VA. These reagents are formulated such that they may be used with most standard oligonucleotide synthesis machines (such as those available from Applied Biosystems, Millipore, or Beckman) and do not require special manipulations or special personnel. Molecules of this general type may be prepared following standard procedures. Example 1

Oligonucleotides bearing intercalating acridine derivatives which are appended to the 5' end of the nucleotide strand can be prepared as follows:

detrttYl.tloπ

Wherein DMT is dimethoxytrityl; Ac₂O is acetic anhydride; and NMI is N-methyl imidazole.

One or more intercalating acridine derivative units may be appended to the 5' end of an oligonucleotide by use of the acridine phosphoramidite reagent (Acr, 1). This reagent is available commercially from Glen Research Corporation, Inc. What is illustrated is the use of the Acr reagent to prepare the modified deoxyoligonucleotide 3'- G-A-C-Acr-Acr-5'. First, the G, A, and C deoxyribose phosphoramidite monomers are coupled sequentially to the solid support in the normal way to give the resin bound trinucleotide (2). The dimethoxyltrityl protecting group on the terminal C is then removed by treatment with dichloroacetic acid (detritylation) to provide alcohol (3). Treatment of alcohol 3 with the Acr reagent (coupling) followed by oxidation with iodine in THF solvent (oxidation) and then acetic anhydride/N-methyl imidazole (capping) joins a single intercalating acridine derivative to the 5 '-end of the trinucleotide via a phosphate triester linkage (4). Additional intercalating acridine derivative molecules may be introduced by repeating the following series of steps: detritylation, coupling with Acr, oxidation, and capping (5). The four carbon alkyl group between the acridine amino and die phosphoro chain serves as a spacer bridge, facilitating spacing of the intercalating moieties. Once the desired number of intercalating acridine derivative molecules have been introduced, the oligonucleotide is cleaved from the solid support by the action of ammonium hydroxide (cleavage) and purified by gel electrophoresis or high performance liquid chromatography. Although for clarity we have applied the Acr reagent to synthesize a modified deoxy trinucleotide, me procedure described above is equally suitable for the synthesis of modified deoxyoligonucleotides containing as many as 60 residues. Moreover, with only minor modification (the use of ribose phosphoramidites as opposed to deoxyribose phosphoramidites) the aforesaid procedure could also be applied to the synthesis of intercalating acridine derivative-modified oligoribonucleotides. Example 2

Oligonucleotides bearing intercalating acridine derivatives appended to d e 3' end. Because solid-phase oligonucleotide synthesis proceeds in the 3' to 5' direction, specially modified solid supports are needed to append intercalating acridine derivatives to die 3' end of a synthetic oligonucleotide. The required acridine-modified controlled pore glass support (acridine CPG, 5) is available from Glen Research Corporation. Example 2 illustrates the use of acridine CPG to prepare the modified oligonucleotide 3'-Acr-Acr-G- A-C-5'. First, acridine CPG 5 is used to initiate oligonucleotide synthesis. The dimethoxytrityl protecting group is removed (detritylation) and a second intercalating acridine derivative is introduced by reaction of d e newly generated hydroxyl group widi the Acr reagent. After die requisite oxidation and capping steps, standard G, A, and C deoxynucleotide phosphoramidites are added sequentially to the growing oligonucleotide chain to provide die resin-bound trinucleotide 6. A final detritylation step followed by chain cleavage provides die desired product. The procedure shown in Example 2 is general and suitable for the synthesis of modified deoxyoligonucleotides containing at least 60 residues. Moreover, widi only minor modification (the use of ribose phosphoramidites as opposed to deoxyribose phosphoramidites) the procedure outlined in Example 2 could be applied to the synthesis of intercalating acridine derivative-modified oligoribonucleotides as well.

In the procedure for the synthesis of oligonucleotides modified at the 3' end with multiple acridine units DMT is dimethoxyltrityl; Ac₂O is acetic anhydride; and NMI is N- methyl imidazole. Example 3

Oligonucleotides bearing intercalating acridine derivatives appended to both the 5' and 3' ends can be prepared by combining the procedures outlined in Examples 1 and 2. Example 4

Oligonucleotides bearing several internal intercalating acridine derivative units may be prepared as follows: Modified oligonucleotides bearing several internal intercalating acridine derivative units may be prepared by substituting phosphoramidite 7 for a standard nucleotide phosphoramidite in any internal coupling step. Phosphoramidite 7 can be prepared easily via die series of steps shown below. The synthesis proposed is based on procedures reported in the chemical literature for closely related molecules and is simple enough to be performed on large scale by a B.S. level chemist or skilled technician. A notable feature of compound 7 is that it retains the Watson-Crick base pairing capabilities of an unmodified cytosine residue:

Wherein DMT-CI is di ethoxytrityl chloride; TBDMS-C1 is tert-bu yl dimethylsilyl chloride; and Et₃N is triethyl amine.

COMBINATION WITH OPTIONAL INGREDIENTS The intercalating oligonucleotides may be compounded with suitable pharmaceutical carriers, diluents and/or other optional ingredients. As an example of such an ingredient other than typical carriers and diluents, the intercalating oligonucleotide might be associated with a lipophilic macromolecule or other type of carrier macromolecule to enhance the ability of the intercalating oligonucleotide to pass through a cell membrane. Such a carrier macromolecule might have a targeting molecule, such as a specific antibody, attached to it. Once such a complex enters the bloodstream, the targeting antibody helps insure that the intercalating oligonucleotide and its carrier macromolecule attach to and penetrate the desired target cell. Yet another alternative might be to attach a targeting antibody directly to an intercalating oligonucleotide sequence.

PHARMACEUTICAL ADMINISTRATION OF THE INTERCALATING OLIGONUCLEOTIDES

Pharmaceutical compositions can be crafted for treating numerous diseases of varying types. Many modes of administration of the intercalating oligonucleotide pharmaceutical agents will be apparent to those of ordinary skill in the art. Intravenous injection to a body water concentration of 5-300 micromolar is one technique. Concentration can be determined based on the fact that body water comprises 60 to 65 % of body weight. Topical application is contemplated. Application to the eye, skin or mucous membranes are exemplary modes of topical application.

Direct injection into a tumor is one possible route for treating some types of cancer. Injection into particular organs or glands to affect activity is possible. Once inside a cell, d e intercalating oligonucleotide would seek out with great specificity the target DNA or RNA segment. In Fig. 10, an oligonucleotide sequence 1, which is complementary to DNA or RNA strand 4 of a double helix comprised of strands 4 and 5, includes parallel intercalating chains 2-12 and 3-13, which are shown schematically interposing themselves between adjacent base pairs hybridizing on strands 4 and 5. In Fig. 11, oligonucleotide strand 6 which is complementary to a single strand of DNA or RNA 9, is illustrated with parallel intercalating agent chains 7 and 17 and 8 and 18 interposed between adjacent base pairs hybridizing strand 6 to strand 9.

TREATMENT OF IN VITRO SAMPLES Referring now to Fig. 12, there is disclosed a typical blood sample drawing receptacle 18 which will have the virus-neutralizing reagents coated onto or otherwise incorporated into the interior 21 thereof. The receptacle may be an evacuated tube of the type sold by Becton Dickinson and Company under me trademark Vacutainer^*. The receptacle 18 has an integral end wall 22 and a rubber stopper 24 which closes an open end of die receptacle 18. Fig. 13 illustrates a blood or blood product storage bag 26 which can have its interior surface dry coated or od erwise incorporated therein with additives. Any compartments attached to d e bag can similarly contain such additives.

In one embodiment, these additives or reagents comprise the complementary synthesized oligonucleotide strands with intercalating agent units attached to them in the manner described above, whereby an additive is formed which, when mixed wid a biological fluid sample such as anticoagulated whole blood, plasma, or serum, or the like, will serve to render a significant number, if not all, of the target viruses in the sample non-infectious. The synthesized oligonucleotide strands will have a monomer sequence which compliments a target monomer sequence in the viral genome or messenger RNA. The intercalating units on the synthetic strands enable the synthetic strands to create localized double or triple-stranded highly stable nucleotide complexes widi the viral RNA or DNA, as applicable, which complexes render the virus incapable of replicating. The risk of becoming infected by exposure to a sample which has been treated with the additive is dius significantly reduced.

In most cases, me sample being tested will be incubated with die test reagents for a period of about 30 minutes, whereupon essentially all of the targets in die sample will have been engaged by the additive, and about 99% or more of d e target infectious agents in the sample will have been rendered non-infectious. While the invention has been described primarily in connection with viruses, it may also be used to retard tumor development which is often associated widi enhanced expression of various oncogenes diat include: abl; fes; erb; ras; myc; and others. Inhibiting transcription and/or translation of the oncogenes by targeting one or more of the aforesaid sequences would allow an appropriate additive to serve as an antitumor agent.

EMBODIMENTS DIRECTED PRIMARILY AGAINST EXTRACELLULAR OR FREE VIRUS In another embodiment of the invention, freely circulating, extracellular viruses such as those causing: AIDS, HIV-1 or HIV-2; various forms of hepatitis, such as hepatitis B virus, hepatitis A virus, and hepatitis C virus; as well as cytomegalic disease, CMV, found in some in vitro samples of whole blood, blood plasma, or blood serum are rendered non-infectious thereby rendering d e samples substantially less infectious, by effectively blocking surface epitopes on the extracellular viral capsids widi one or more binding agents directed against such surace epitopes. The circulating pathogenic viral capsids therefore cannot bind to dieir respective target blood cells. This embodiment of the invention mimics one way that the mammalian body neutralizes previously experienced viruses from causing subsequent infection.

The aforesaid surface epitope-blocking is accomplished by providing a viral capsid epitope-specific binding agent in the additive, which binding agent may be a monoclonal antibody; a naturally produced polyclonal antibody; a multitude of different monoclonal and/or polyclonal antibodies; and/or one or more oligonucleotides diat have the property of binding an antigen with ie same specificity as do protein antibodies, i.e., an aptamer. The binding agent or agents is commingled widi the blood sample as, or after, the latter is drawn. As noted, d ese binding agents may be immunoglobulin, eidier monoclonal or polyclonal, and/or oligonucleotides whose tertiary configuration is chosen, by methods described by Nexagen, a public corporation located in Boulder, Colorado specializing in the selection and production of aptamers for therapeutic and diagnostic use. The binding agents afford a high degree of binding widi viral capsid epitopes which are specific to target blood cells, such as lymphocytes, monocytes, reticulocytes or odier cells such as liver cells or brain cells to which the viral capsid can bind, and merefore can infect. The capsid epitope-binding additive tiius occupies target cell "docking sites" on the virus capsid, and severely retards ie ability of the virus to bind to host cells. This significantly lowers die probability that die host sample will infect any body exposed diereto. The aptamers used in die invention can be produced by means of die polymerase chain reaction (PCR) process or other chemical synthesis, coupled widi reverse transcription of RNA or by other means of nucleotide amplification.

The capsids of viruses have many copies of one or more surface epitopes which are specific to their mammalian target cells. These epitopes enable the viral capsids to bond to the target cells. The target cells could be, for example, blood cells, liver cells, respiratory epithelium cells, brain cells, or die like depending on me virus in question. The virus infects the target cells to which the viral capsids have attached, by invading die target cells with viral genetic material contained widiin the capsid. In die HIV capsid, die target blood cell-specific epitope can be die gp 120 glycoprotein epitope. Viral capsid epitopes provide a means which enables the capsid to attach to, and diereby infect mammalian cells.

In order to interfere with the ability of freely circulating HTV viruses contained in a blood or biological sample from binding to, and thereby infecting targeted cells in one exposed to an infected blood sample, an epitope binding agent is added to d e blood sample, which epitope binding agent is specific to target cell epitopes on the viral capsid. The antibody or other binding agent will attach to the target epitopes on any freely circulating viral capsids in d e blood sample to a degree which neutralizes such target epitopes, thus rendering d e viral capsids incapable of attaching to target cells. Sufficient binding agent will be introduced into the blood sample to ensure neutralization of all freely circulating target virus capsids in die sample. The binding agent for the HIV capsid can be a monoclonal or polyclonal antibody, or an aptamer having a strong affinity for the cell-specific epitopes on the viral capsid. The binding agent can be specific to gp 120 glycoprotein epitopes on me HIV viral capsid. A binding agent for hepatitis viral capsids can be specific to d e hepatitis B surface antigen epitope or its subdivisions on d e hepatitis viral capsid. More than one type of binding agent per virus may be used to ensure binding in die presence of genetic diversity manifested by epitopic variations. When the binding agent is an aptamer having a strong affinity for the cell-specific epitopes on the viral capsid, d e specific oligonucleotide can be mass produced by polymerase chain reaction (PCR) technology coupled wid reverse transcription of RNA.

Fig. 14 is illustrative of the manner of operation of the extracellular embodiment of die invention. The viral capsid is denoted by the numeral 12, and d e binding particles are denoted by die numeral 14. Each cellular epitope-specific site 16 on the viral capsid 12 has a binding unit attached diereto so diat there are no free binding sites 16 on the capsid. The capsid 12 therefore cannot bind to a target cell and thus cannot infect one who is exposed to a sample containing the neutralized viral capsid. Of course, it is understood diat die foregoing is merely a preferred embodiment of the invention and mat various changes and alterations can be made without departing from the spirit and broader aspects thereof as defined by the appended claims, which are to be interpreted in accordance with d e principles of patent law.

Claims

The embodiments of d e invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound comprising an oligonucleotide sequence complementary to a targeted sequence on the genome or RNA of an organism or virus, having a plurality of intercalating molecules attached diereto.

2. The compound of claim 1 in which said intercalating molecules are attached to said oligonucleotide sequence in series, in that they are attached to one another and one of d em is attached to said oligonucleotide sequence at one location on said oligonucleotide sequence.

3. The compound of claim 2 in which said oligonucleotide sequence is comprised of 15 to 50 nucleotides.

4. The compound of claim 2 in which said oligonucleotide sequence is comprised of 15 to 30 nucleotides.

5. The compound of claim 2 in which said oligonucleotide sequence is comprised of 20 to 28 nucleotides.

6. The compound of claim 5 in which said intercalating molecules are selected from die group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, anthracycline compounds, benzo (e) pyridol indole, and combinations thereof.

7. The compound of claim 5 in which said intercalating molecules are selected from the group consisting of acridine and acridine derivatives.

8. The compound of claim 3 in which said intercalating molecules are selected from the group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ediidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, andiracycline compounds, benzo (e) pyridol indole, and combinations mereof.

9. The compound of claim 3 in which said intercalating molecules are selected from the group consisting of acridine and acridine derivatives.

10. The compound of claim 3 in which said intercalating molecules in any series are spaced by spacer bridges such at adjacent intercalating molecules are spaced sufficiently diat they can insert between spaced, non-adjacent base pairs.

11. The compound of claim 10 in which said spacer bridges are aliphatic groups.

12. The compound of claim 11 in which said aliphatic groups comprise 3-5 carbons.

13. The compound of claim 1 in which said intercalating molecules are selected from me group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, andiracycline compounds, benzo (e) pyridol indole, and combinations thereof.

14. The compound of claim 1 in which said intercalating molecules are selected from e group consisting of acridine and acridine derivatives.

15. The compound of claim 1 in which said intercalating molecules are attached in parallel, in that each is attached to said oligonucleotide at a different location.

16. The compound of claim 15 in which said oligonucleotide sequence is comprised of 15 to 50 nucleotides.

17. The compound of claim 15 in which said oligonucleotide sequence is comprised of 15 to 30 nucleotides.

18. The compound of claim 15 in which said oligonucleotide sequence is comprised of 20 to 28 nucleotides.

19. The compound of claim 18 in which said intercalating molecules are selected from the group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, andiracycline compounds, benzo (e) pyridol indole, and combinations mereof.

20. The compound of claim 18 in which said intercalating molecules are selected from the group consisting of acridine and acridine derivatives.

21. The compound of claim 16 in which said intercalating molecules are selected from the group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, anthracycline compounds, benzo (e) pyridol indole, and combinations thereof.

22. The compound of claim 16 in which said intercalating molecules are selected from me group consisting of acridine and acridine derivatives.

23. The compound of claim 16 in which any two of said parallel intercalating molecules are joined to nucleotides which are separated on said oligonucleotide segment by at least one intervening nucleotide, such diat said parallel intercalating agents can insert between non-adjacent base pairs.

24. The compound of claim 1 in which said intercalating molecules are attached bodi in series and in parallel, in that at least two intercalating molecules are attached to each other, and one of d em is attached to said oligonucleotide sequence at a single location; and at least one odier intercalating molecule is attached to said oligonucleotide sequence at a different location.

25. The compound of claim 24 in which said oligonucleotide sequence is comprised of 15 to 50 nucleotides.

26. The compound of claim 24 in which said oligonucleotide sequence is comprised of 15 to 30 nucleotides.

27. The compound of claim 24 in which said oligonucleotide sequence is comprised of 20 to 28 nucleotides.

28. The compound of claim 27 in which said intercalating molecules are selected from the group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, andiracycline compounds, benzo (e) pyridol indole, and combinations mereof.

29. The compound of claim 27 in which said intercalating molecules are selected from the group consisting of acridine and acridine derivatives.

30. The compound of claim 25 in which said intercalating molecules are selected from me group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, anthracycline compounds, benzo (e) pyridol indole, and combinations thereof.

31. The compound of claim 25 in which said intercalating molecules are selected from die group consisting of acridine and acridine derivatives.

32. The compound of claim 25 in which said intercalating molecules in any series are spaced by spacer bridges, and said parallel intercalating molecules are attached to nucleotides which are separated by at least one intervening nucleotide, such diat adjacent intercalating molecules are spaced sufficiently d at diey can insert between spaced, non- adjacent base pairs.

33. The compound of claim 32 in which said spacer bridges are aliphatic groups.

34. The compound of claim 33 in which said aliphatic groups comprise 3-5 carbons.

35. The compound of claim 1 in which at least two intercalating molecules are attached to each other, and one of them is attached to said oligonucleotide sequence at die 3' end d ereof.

36. The compound of claim 1 in which at least two intercalating molecules are attached to each other, and one of d em is attached to said oligonucleotide sequence at die 5' end diereof.

37. The compound of claim 1 in which at least two oligonucleotide molecules are attached to each odier, and one of them is attached to said oligonucleotide sequence between die 3' and 5' ends diereof.

38. The compound of claim 1 in which at least one intercalating molecule is attached to each of d e 3' and 5' ends of said oligonucleotide sequence.

39. The compound of claim 1 in which at least one intercalating molecule is attached to either d e 3' or 5' end of said oligonucleotide sequence, and at least one other is attached to said oligonucleotide sequence between d e 3' and 5' ends thereof.

40. The compound of claim 1 in which said oligonucleotide sequence is modified to resist enzymatic degradation.

41. The compound of claim 40 in which said oligonucleotide sequence is comprised of 15 to 50 nucleotides.

42. The compound of claim 41 in which said intercalating molecules are selected from the group consisting of acridine and its derivatives, oxazolopyridocarbazole chromophore, mitoxantrone, ethidium derivatives which are capable of penetrating live cells, ellipticine derivatives, sterol dyes, andiracycline compounds, benzo (e) pyridol indole, and combinations diereof.

43. The compound of claim 40 in which said intercalating molecules are attached to said oligonucleotide sequence in series, in that they are attached to one anodier and one of d em is attached to said oligonucleotide sequence at one location on said oligonucleotide sequence.

44. The compound of claim 40 in which said aliphatic groups comprise 3-5 carbons.

45. The compound of claim 40 in which said intercalating molecules are selected from the group consisting of acridine and acridine derivatives.

46. The compound of claim 40 in which said oligonucleotide sequence is modified to comprise a thiodiester backbone.

47. The compound of claim 1 in which said intercalating molecules are attached to said oligonucleotide sequence in series, in that they are attached to one anodier and one of d em is attached to said oligonucleotide sequence at one location on said oligonucleotide sequence; in which said intercalating molecules in any series are spaced by spacer bridges such d at adjacent intercalating molecules are spaced sufficiently diat they can insert between spaced, non-adjacent base pairs.

48. The compound of claim 1 in which said intercalating molecules are attached in parallel, in that each is attached to said oligonucleotide at a different location; in which any two of said parallel intercalating molecules are joined to nucleotides which are separated on said oligonucleotide segment by at least one intervening nucleotide, such that said parallel intercalating agents can insert between non-adjacent base pairs.

49. A pharmaceutical agent comprising: an oligonucleotide sequence complementary to a targeted sequence on d e genome or RNA of an organism or virus, having a plurality of intercalating molecules attached diereto, combined with a pharmaceutical carrier or diluent.

50. A method for treating or preventing disease comprising: identifying a DNA or RNA sequence on a disease cell, a disease causing organism or a disease causing virus, or an RNA or DNA sequence capable of producing a protein which inhibits the production of desirable protein or an RNA or DNA sequence coding for protein which facilitates reproduction of a disease cell, or an RNA or DNA sequence which produces a protein which causes or enables causation of a disease; preparing an oligonucleotide sequence complementary to said target sequence; attaching a plurality of intercalating molecules to said complementary oligonucleotide sequence; and administering the resulting intercalating oligonucleotide to a subject to be treated.

51. A synthetic additive for incorporation into a biological sample, said additive having ingredient units which comprise synthetic strands of oligonucleotides, and/or modified nuclease-resistant derivatives of oligonucleotides, which will impair genetic expression in viral and/or mammalian targets, said strands having intercalating agents attached diereto which enhance the strengdi of complexes formed by d e syndietic strands wid genetic material in the targets, and said strands being operable to form highly stable complexes with target genomes or target messenger RNA, said complexes being of sufficient stability to prevent said target genomes from replicating and/or transcribing, and/or to prevent said target messenger RNA from being translated into protein.

52. The additive of claim 51 wherein said syndietic strands include a base monomer sequence which complements a unique monomer sequence on the target genome or messenger RNA, and which base monomer sequence is sufficiently long to ensure formation of the stable complex at said target monomer sequence, and to minimize d e formation of complexes at random locations on the target genome or messenger RNA due to random matching of the syndietic strands widi shorter nontarget sequences on die target genome or messenger RNA.

53. The additive of claim 52 wherein said syndietic strands include a plurality of separate intercalating agent units which are attached to die syndietic strands on at least two different internal sites on said strands.

54. The additive of claim 52 wherein said syndietic strands include at least one intercalating agent chain which is composed of a sequence of interconnected intercalating agent molecules, which intercalating agent chain is attached to a terminus site on d e syndietic strands.

55. The additive of claim 54 wherein said intercalating agent unit is attached to bodi terminus sites on the syndietic strands.

56. The additive of claim 52 wherein said syndietic strands comprise between about 20 to about 28 base monomers.

57. The additive of claim 51 wherein said syndietic strands include intercalating agents selected from the group consisting of: intercalating acridine derivatives; oxazolopyridocarbazole chromophore (OPC); mitoxantrone; ethidium derivatives which are capable of penetrating live cells; sterol dyes; andiracycline compounds such as doxorubicin; benzo (_*= ) pyridol indol; and/or groove binders selected from the group consisting of: Hoechst-33258; and diiazole yellow and derivatives diereof.

58. A method for impairing genetic expression in viral and/or mammalian targets contained in a biological sample, said method comprising the step of incubating the sample with an additive which contains syndietic strands of oligonucleotides, and/or modified nuclease-resistant derivatives of oligonucleotides which will impair target genetic expression and which complement at least one localized target monomer sequence found on target genomes and/or messenger RNA, said syndietic strands having intercalating agents attached diereto which enhance the strength of complexes formed by d e syndietic strands with genetic material in the targets, said syndietic strands being operative to penetrate targets to form highly stable complexes with said target genomes and/or messenger RNA, which complexes are sufficiendy stable to prevent said target genomes from replicating and to prevent said target messenger RNA from being translated into protein.

59. The method of claim 58 wherein said additive strands include a base monomer sequence which complements at least one target monomer sequence on the target genomes or messenger RNA, and which base monomer sequence is sufficiently long to ensure formation of me stable complex at said target monomer sequence, and to minimize the formation of complexes at random locations on the target genomes or messenger RNA due to random matching of the additive strands widi shorter nontarget sequences on die target genomes or messenger RNA.

60. The method of claim 59 wherein said additive strands include a plurality of separate intercalating agent units which are attached to die additive strands on at least two different internal sites on the strands.

61. The method of claim 59 wherein said additive strands include at least one intercalating agent chain which is composed of a sequence of interconnected intercalating agent molecules, which intercalating agent chain is attached to a terminus site on the additive strands.

62. The method of claim 61 wherein intercalating agent chains are attached to bo terminus sites on die additive strands.

63. The method of claim 58 wherein said additive strands comprise between about 20 to about 28 base monomers.

64. The method of claim 58 wherein said additive strands include intercalating agent ingredients which are formed from planar aromatic compounds include intercalating agents selected from the group consisting of: intercalating acridine derivatives; oxazolopyridocarbazole chromophore (OPC); mitoxantrone; edύdium derivatives which are capable of penetrating live cells; sterol dyes; andiracycline compounds such as doxorubicin; benzo (e ) pyridol indol; and/or groove binders selected from the group consisting of: Hoechst-33258; and diiazole yellow and derivatives diereof.

65. The method of claim 58 wherein said additive strands include:

(a) a first plurality of strands having a base monomer sequence which complements a first target monomer sequence on the target genomes or messenger RNA, and which first base monomer sequence is sufficiently long to ensure formation of me stable complex at said first target monomer sequence, and to minimize die formation of complexes at random location on die target genomes or messenger RNA due to random matching of the additive strands with shorter nontarget sequences on the target genomes or messenger RNA; and

(b) a second plurality of strands having a base monomer sequence which complements a second target monomer sequence on the target genomes or messenger RNA, and which second base monomer sequence is sufficiently long to ensure formation of the stable complex at said second target monomer sequence, and to minimize the formation of complexes at random locations on die target genomes or messenger RNA due to random matching of die additive strands widi shorter nontarget sequences on die target genomes or messenger RNA.

66. A syndietic additive possessing an avidity against at least one target monomer sequence in objective target genetic strands found in viruses, or in mammalian biological cells, said additive having ingredients which will impair impression of the objective target genetic strands, said additive comprising: synmesized strands of oligonucleotides, and/or modified nuclease-resistant derivatives of oligonucleotides, said synd esized strands having intercalating agents attached diereto which enhance the strength of complexes formed by d e syndiesized strands widi genetic material in the target genetic strands, said syndiesized strands being operative to form at least one localized complex on a complement target genetic sequence in the subjective target genetic strands, said localized complex being sufficiently stable so as to prevent any such complexed intracellular genomes from replicating, and to prevent any such complexed defined intracellular messenger RNA from being translated into protein.

67. The additive of claim 66 wherein said syndiesized strands are sufficiently long to ensure formation of the stable complex at said target genetic monomer sequence, and to minimize die formation of complexes at random locations on die objective genetic material which could result from random matching of die oligonucleotide/intercalating agent strands wid shorter sequences on die objective genetic material.

68. The additive of claim 66 wherein said synthesized strands include a plurality of separate intercalating gent units which are attached to said strands on at least two different internal sites on said strands.

69. The additive of claim 68 wherein said strands include at least one intercalating agent chain which is composed of a sequence of interconnected intercalating agent units, which intercalating agent chain is attached to one of die 3' and 5' terminal sites on said strands.

70. The additive of claim 69 wherein an intercalating agent chain is attached to botii 3' and 5' terminal sites on said strands.

71. The additive of claim 69 wherein said strands include intercalating agent molecules which are formed from intercalating acridine derivatives: etiiidium derivatives which are capable of penetrating live cells; anthracycline compounds such as doxorubicin; benzo (e ) pyridol indol (BePi); and/or any related odier planar aromatic or heterocyclic intercalating agents or derivatives diereof.