EP1349959A2

EP1349959A2 - Assay to detect integration of a retrovirus polynucleotide into a target nucleic acid

Info

Publication number: EP1349959A2
Application number: EP01977718A
Authority: EP
Inventors: Richard S. Kornbluth; Alexei Brooun
Original assignee: University of California
Current assignee: University of California
Priority date: 2000-10-10
Filing date: 2001-10-09
Publication date: 2003-10-08
Also published as: AU2001296814A1; CA2424998A1; WO2002031204A2; WO2002031204A3

Abstract

Methods for detecting integration of a polynucleotide such as a retrovirus into a target nucleic acid molecule are provided, as are methods of identifying cellular factors involved in such an integration event and of identifying agents that can modulate such integration events. Also provided are reagents and kits for performing such methods.

Description

NUCLEIC ACID INTEGRATION ASSAY

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION This invention relates generally to integration of mobile genetic elements into nucleic acid molecules, and more specifically to compositions and methods useful for detecting integration of a polynucleotide into a target nucleic acid molecule, for identifying host cell factors involved in such integration, and for identifying agents that modulate such integration.

BACKGROUND INFORMATION

Gene therapy provides a promising means to treat various diseases, including congenital diseases and cancers, by treating the basic genetic defect causing the disease. Essentially, gene therapy involves correcting or substituting for a mutant, deleted or inactive gene in a diseased cell by introducing a normal version of the gene into the cell. As a result, individuals that are successfully treated by gene therapy may be able to cease taking medications or receiving treatments that otherwise would be only palliative, and should experience a vastly improved quality of life.

Gene therapy generally utilizes a viral vector for introducing a gene of interest into the cells of an individual. Viral vectors, which are based on modified forms of viruses, take advantage of the efficiency of viruses to infect cells. In constructing a viral vector, a viral genome is modified by deleting viral genes and introducing a gene of interest, to produce a viral vector genome. A viral vector is produced by propagating the vector genome to generate a mature virus-hke particle containing the vector genome. As such, there is no need to perform separate steps of preparing the gene of interest and incorporating it into the viral vector.

Viral vectors derived from retroviruses have been the most extensively studied. In general, the retroviruses are modified such that genes required for replication of the viral genome and for packaging of the genome into a viral particle are deleted from the viral genome, and genes of interest are inserted in their place. Nevertheless, the viral vector generally has features characteristic of the parent virus, from which the viral vector is derived. For example, retroviruses generally require that a cell divide in order for viral infection to be successful. As such, retroviral vectors generally are useful for introducing a gene of interest into cells that are mitotically active cells or can be induced to divide.

Despite the potential usefulness of viral vectors for delivering a gene of interest into a cell, various problems, often related to the characteristics of the virus from which the vector is derived, remain. For example, a characteristic of retroviruses is that they can integrate into any of a large number of potential target sites in a cellular genome. Thus, while for any particular retrovirus there appear to be "hot spots" for integration, such hot spots are numerous and include nucleotide sequences within genes that are vital to the health and survival of the cells.

In addition to viruses, various other polynucleotides have an ability to integrate into genomic DNA. Such polynucleotides, referred to generally as mobile genetic elements or transposable elements, exist in prokaryotic and eukaryotic organisms, including yeast, plants, insects, and animals. In the human genome, for example, integration events are associated with regions of active genes such in the immunoglobulin and T cell receptor genes, which undergo rearrangements. In the case of T cell receptor genes, for example, the rearrangements mediated by the mobile genetic elements can contribute to the diversity of an immune response. In addition, regions of the human genome that appear to be relatively inactive contain polynucleotide sequences (retroelements) that have characteristics of integrating polynucleotides. Movement of such elements can result in genetic changes that may be beneficial or, more likely, detrimental to an organism.

An understanding of the factors involved in the integration of a polynucleotide into genomic DNA can allow methods to be developed for manipulating such integration. Such methods can be particularly useful, for example, in advancing methods of gene therapy that utilize a retroviral vector, since they may provide a means to prevent random integration of the vector into the genome or to minimize the likelihood of integration into a critical gene. An in vitro assay that accurately represent integration as it occurs in vivo would provide a useful tool for identifying the factors involved in the integration of a polynucleotide such a retrovirus into genomic DNA, and further would provide a convenient system for manipulating the factors involved in integration. Unfortunately, such an assay system, particularly one that can be readily adapted to a high throughput assay format, is not available. Thus, a need exists for an in vitro assay system that accurately represents nucleic acid integration events. The present invention satisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

The present invention relates to a method of detecting integration of a polynucleotide into a target nucleic acid molecule. Such a method can be performed, for example, by contacting a sample, including an integrating polynucleotide and a target nucleic acid molecule, undergo conditions that allow integration of a polynucleotide into the target nucleic acid molecule; and detecting covalent linkage of the integrating polynucleotide and the target nucleic acid molecule. The integrating polynucleotide can be any synthetic or naturally occurring polynucleotide, or portion thereof, that can integrate into a target nucleic acid molecule, generally a double stranded deoxyribonucleic acid molecule, including polynucleotides that integrate in a site-specific manner and polynucleotides that integrate more randomly.

An integrating polynucleotide that can be examined according to a method of the invention can be a viral polynucleotide, a transposon, a retroelement, or any other polynucleotide that can integrate into a target nucleic acid molecule. As such, an integrating polynucleotide can be a polynucleotide sequence of a retrovirus, for example, a retrovirus genome, including a lentivirus genome, for example, a human immunodeficiency virus (HIV) genome such as a genome of HIV-1 or HIV-2, or can be a polynucleotide portion of such a retrovirus genome, including a long terminal repeat (LTR), or nucleotide sequences thereof that are necessary and sufficient for integration, or any portion of a retrovirus genome that has integration activity. An integrating polynucleotide also can be a transposable element such as a yeast transposon, a plant transposon, a Drosophila P element, and the like, including at least the nucleotide sequence of one or both termini having integration activity. In addition, an integrating polynucleotide can be a retroelement as occurs in a eukaryotic genome, particularly a vertebrate genome such as a mammalian genome. Integrating polynucleotides also are exemplified by nucleotide sequences of mammalian genes such as immunoglobulin superfamily genes, which undergo rearrangements.

Accordingly, an integrating polynucleotide can include any nucleotide sequence, provided the polynucleotide includes nucleotide sequences involved in integration such as those present at the termini of naturally occurring integrating polynucleotides.

The integrating polynucleotide can be included in an integration assay of the invention as a discrete component, in which case additional factors required for integration, particularly an integrase, are added to the assay, or the polynucleotide can be associated with one or more factors involved in integration, including, for example, an integrase, or one or more cellular factors, or combinations thereof. In one embodiment, the integrating polynucleotide is a component of a pre-integration complex, which can be obtained, for example, from a retrovirus infected cell. In another embodiment, the integrating polynucleotide comprises a salt stripped pre-integration complex, such a polynucleotide being useful, for example, for identifying cellular factor involved in integration.

The target nucleic acid molecule can include any nucleotide sequence into which an integrating polynucleotide can integrate, including, for example, a randomly generated nucleotide sequence, which can contain two or more different nucleotides; a coding sequence, intronic sequence, or a nucleotide sequence generally found upstream or downstream of a coding sequence in a genome; a repetitive nucleotide sequence such as a moderately repetitive or highly repetitive sequence, as is generally present in a mammalian or other vertebrate or eukaryotic genome; a homopolymeric nucleotide sequence containing a repeat of a single nucleotide such as a polyadenosine sequence, oligothymidine sequence, polyuridine sequence, or the like; or a nucleotide sequence containing a repeat of alternating nucleotide such as poly(dTdA) or poly(dTdC), or the like; or any other nucleotide sequence, particularly where the integrating polynucleotide does not integrate in a site specific (sequence specific) manner. A target nucleic acid molecule can be double stranded or single stranded DNA or RNA, or a DNA:RNA hybrid.

As disclosed herein, a target nucleic acid molecule can be a monomeric target nucleic acid molecule that is about 10 to 1000 nucleotides in length, usually about 50 to 500 nucleotides in length, and particularly about 100 to 250 nucleotides in length. Such target nucleic acid molecules, which provide an effective substrate for integration, are particularly amenable to examination using an amplification reaction such as a polymerase chain reaction (PCR), thus providing a means to detect an integration event. A target nucleic acid molecule also can be constructed as a concatemer of such monomeric target nucleic acid molecules, for example, a concatemer containing about 2 to 1000 monomeric target nucleic acid molecules; generally about 10 to 250 monomeric target nucleic acid molecules, and particularly about 25 to 100 monomeric target nucleic acid molecules. As disclosed herein, a concatemerized target nucleic acid molecule provides a particularly effective substrate for integration, and further provides a target in which integration events conveniently can be detected by an amplification reaction such as PCR.

According to a method of the invention, detecting covalent linkage of an integrating polynucleotide to the target nucleic acid molecule can be performed in any of various ways. For example, such covalent linkage can be detected by contacting the sample with an exonuclease having 5' exonuclease activity, thereafter contacting the sample with at least one primer that selectively hybridizes at or near the 3' terminus of the target nucleic acid molecule, under conditions that allow primer extension fiom the oligonucleotide primer; and detecting generation of the primer extension product, or under conditions that allow linear amplification from the oligonucleotide primer, and detecting generation of the linear amplification product. The primer extension or linear amplification product can be detected, for example, by further contacting the sample with an amplification primer pair, which includes a first primer that selectively hybridizes to a nucleotide sequence of the integrating polynucleotide, and a second primer that selectively hybridizes to a nucleotide sequence of the primer extension or linear amplification product, under conditions that allow generation of an amplification product from the amplification primer pair; and detecting generation of the amplification product. In one embodiment, the conditions are such that melting temperature is below that which allows selective hybridization of the primers of the amplification primer pair, and is above that which allows selective hybridization of the oligonucleotide primer used for the linear amplification reaction. In another embodiment, the oligonucleotide primer is designed such that it can form a secondary structure, which can prevent the oligonucleotide primer from selectively hybridizing at or near the 3' terminus of the target nucleic acid molecule, and the conditions that allow generation of the amplification product comprise conditions below the melting temperature of a secondary structure formed by the oligonucleotide primer.

Detecting covalent linkage of an integrating polynucleotide and the target nucleic acid molecule also can be performed by contacting the sample with a first amplification primer pair, which includes a first primer that selectively hybridizes to a nucleotide sequence of the integrating polynucleotide, and a second primer that selectively hybridizes to a nucleotide sequence of the target nucleic acid molecule, under conditions that allow generation of a first amplification product from the primer pair; and detecting generation of an amplification product. The amplification product that is detected can be the first amplification product; or the method can further include contacting the sample with a detector oligonucleotide, which can selectively hybridize to a nucleotide sequence of the first amplification product; and detecting selective hybridization of the detector oligonucleotide to the first amplification product; or can further include contacting the sample with a bilabeled oligonucleotide probe such as a molecular beacon or a TaqMan™ probe, which includes a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the first amplification product; and detecting fluorescence due to unquenching of the fluorescent moiety.

The amplification product that is detected also can be a second amplification product, in which case the method further includes contacting the sample with at least a first primer of a second amplification primer pair, wherein the first primer of the second amplification primer pair can selectively hybridize to a nucleotide sequence of the first amplification product, under conditions that, in the presence of a second primer of the second amplification primer pair, allow generation of a second amplification product; and generating a second amplification product. The first primer or second primer of the first amplification primer pair can function as the second primer of the second amplification primer pair, or the method can further include contacting the sample with the second primer of the second amplification primer pair.

In one embodiment, a method of detecting integration of a polynucleotide into a target nucleic acid molecule can be performed, for example, by contacting a sample with a pre-integration complex (PIC), which includes an integrating polynucleotide and an integrase, and the target nucleic acid molecule, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule; thereafter decreasing the effective concentration of target nucleic acid molecules in the sample; further contacting the sample with a first amplification primer pair, under conditions that allow generation of a first amplification product from an integration product of the integrating polynucleotide of the PIC and the target nucleic acid molecule, and detecting generation of an amplification product, thereby detecting integration of the polynucleotide into the target nucleic acid molecule. The first amplification primer pair can include, for example, a first primer that selectively hybridizes to a nucleotide sequence of the integrating polynucleotide of the PIC, and a second primer that selectively hybridizes to a nucleotide sequence of the target nucleic acid molecule. The effective concentration of target nucleic acid molecules in a sample can be decreased, for example, diluting the sample; by contacting the sample with an exonuclease that degrades unreacted target nucleic acid molecules; by diluting the sample and contacting it with an exonuclease that degrades unreacted target nucleic acid molecules; and the like.

The step of detecting generation of the amplification product can be performed by further contacting the sample with a bilabeled oligonucleotide probe such as a TaqMan™ probe, which includes a fluorescent moiety and a fluorescent quencher, wherein the oligonucleotide probe selectively hybridizes to a nucleotide sequence of the polynucleotide downstream from the first amplification primer, or a nucleotide sequence of the target nucleic acid molecule downstream rom the second amplification primer; and detecting fluorescence due to the fluorescent moiety. The step of detecting generation of an amplification product also can be performed by contacting the first amplification with at least a first primer of a second amplification primer pair, under conditions that, in the presence of a second primer of the second amplification primer pair, allow generation of a second amplification product, wherein the first primer of the second amplification primer pair selectively hybridizes to a nucleotide sequence of the first amplification product; and detecting generation of the second amplification product. The second primer of the second amplification primer pair can be one of the first primer or second primer of the first amplification pair, wherein the second amplification product is a hemi-nested amplification product; or can be a second primer that selectively hybridizes to'a nucleotide sequence of the first amplification product, wherein the second amplification product is a nested amplification product.

The present invention also relates to a method of identifying an agent that modulates integration of a polynucleotide into a target nucleic acid molecule. Such a method can be performed, for example, by contacting the polynucleotide, the target nucleic acid molecule, and a test agent, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule; and detecting a change in the number of integration events of the polynucleotide and the target nucleic acid molecule in the presence of the test agent as compared to the number of integration events in the absence of the test agent, wherein a change identifies the test agent as an agent that modulates integration of the polynucleotide into the target nucleic acid molecule. The number of integration events can be detected after the polynucleotide, target nucleic acid molecule, and test agent have been contacted for a predetermined period time, or the number of integration events occurring per unit period of time can be determined over a period of time, thereby providing a means to identify an agent that modulates the rate of integration of a polynucleotide into a target nucleic acid molecule. An agent that can reduce or inhibit the number of integration events effected by an integrating polynucleotide or that can increase the number of integration events can be any type of molecule, including, for example, a peptide; a polynucleotide; a derivative of a peptide or polynucleotide such as a peptide nucleic acid, which is a nucleic acid molecule containing one or more peptide bonds linking the nucleotide monomers; a small organic molecule such as a peptidomimetic; and the like. In one embodiment, the test agent comprises one of a library of test agents, in which case the method can, but need not, be performed in a high throughput format. The library of test agents can be any type of library, for example, a library of randomly generated, biased, or variegated molecules.

The present invention further relates to a method of identifying a factor that mediates integration of a polynucleotide into a target nucleic acid molecule. Such a method can be performed, for example, by contacting the polynucleotide and the target nucleic acid molecule with a test factor, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule; and detecting integration of the polynucleotide into the target nucleic acid molecule. The test factor can be a synthetic molecule such as a peptide, polynucleotide, or the like, which can be one of a library of molecules; or can be naturally occurring, for example, a cellular factor such as a cellular protein, proteolipid, glycoprotein, polynucleotide, nucleoprotein, or the like. A method of identifying a factor that mediates integration of a polynucleotide into a target nucleic acid molecule can further include a step of isolating the factor. Accordingly, the present invention provides an isolated factor obtained using such a method.

The present invention relates to a method of generating a linear amplification product of a selected strand of a nucleic acid molecule. Such a method can be performed, for example, by contacting a sample containing the nucleic acid molecule with at least one oligonucleotide primer that selectively hybridizes to the selected strand, under conditions that allow generation of a linear amplification product comprising the oligonucleotide primer, wherein the oligonucleotide primer can form a secondary structure, which prevents the primer from selectively hybridizing to the selected strand, and wherein the conditions that allow generation of the linear amplification product include conditions above the melting temperature of the secondary structure of the oligonucleotide primer.

The oligonucleotide primer can selectively hybridize at any position on the selected strand, provided that extension of the primer and linear amplification can occur, particularly at or near a 3' terminus of the nucleic acid molecule. The nucleic acid molecule can be single stranded, for example, deoxyribonucleic acid strand complementary to an mRNA (i.e., a cDNA), in which case the single strand is the selected strand, or can be a double stranded nucleic acid molecule, which includes a first strand and a second strand, either of which can be the selected strand for linear amplification. For example, the nucleic acid molecule can be a double stranded DNA which contains a nick in one strand, wherein, in a method of the invention, the second strand is the selected strand, and the linear amplification product generated therefrom spans the nick. In one embodiment, the selected strand includes an integration junction, which is formed by integration of a polynucleotide into a target nucleic acid molecule.

The method of generating a linear amplification product can further include using the linear amplification product to generate an exponential amplification product. Such a method can be performed, for example, by contacting the linear amplification product with an amplification primer pair, which includes a first primer that selectively hybridizes to a nucleotide sequence of the selected strand of the nucleic acid molecule, and a second primer that selectively hybridizes to a nucleotide sequence of the linear amplification product, under conditions that allow generation of an amplification product from the amplification primer pair, wherein such conditions include conditions that are below the melting temperature of a secondary structure formed by the oligonucleotide primer, thereby preventing the oligonucleotide primer from selectively hybridizing to the selected strand. The method of generating a linear amplification product can further include detecting generation of the linear amplification product. Such detecting can be performed, for example, by contacting the sample with a bilabeled oligonucleotide probe, which comprises a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the selected strand that is in a 3' position with respect to the oligonucleotide primer; and detecting fluorescence due to the fluorescent moiety. Such a detecting method similarly can be used to detect generation of an amplification product, in a method in which the linear amplification product is used as a template for an amplification reaction such as PCR, for example, by contacting the sample containing the amplification primer pair with a bilabeled oligonucleotide probe, which can selectively hybridize to a nucleotide sequence of the amplification product; and detecting fluorescence due to the fluorescent moiety.

The nucleic acid molecule containing the selected strand can be an isolated nucleic acid molecule that is added to a reaction mixture for performing a method of the invention. The sample, which contains the nucleic acid molecule, also can be all or a portion of a naturally occurring sample, for example, a cell sample, which is obtained from a subject using a method such as a biopsy procedure. The subject can be any subject for which it is desired to generate a linear amplification product of a selected strand of a nucleic acid molecule, which can be a nucleic acid molecule normally present in the cell such as a portion of genomic DNA or plasmid DNA, or that can be derived from a naturally occurring nucleic acid molecule, for example, a cDNA derived from an mRNA. Generally, the subject is a vertebrate subject such as a mammalian subject, for example, a human subject.

A method of the invention provides a means to obtain a linear amplification product of a selected strand of a nucleic acid molecule. As such, the method can be used to enrich a mixed population of nucleic acid molecules for one or a family of related nucleic acid molecules, or for obtaining a relatively large amount of the selected strand from a sample containing a relatively small amount of the nucleic acid molecule containing the selected strand. For example, the oligonucleotide primer used in the method can be designed so as to selectively hybridize to a conserved nucleotide sequence that is common to a population of related but different nucleic acid molecules such as nucleic acid molecules that encode a polypeptide domain or a peptide portion thereof, or nucleic acid molecules that encode a signal peptide.

One or more linear amplification products generated according to a method of the invention can be further contacted with a hybridization probe, which can selectively hybridize to a nucleotide sequence of the linear amplification product, such hybridizing being useful for detecting or isolating the linear amplification products. In one embodiment, the hybridization probe is immobilized to a solid support, which provides a convenient means to isolate the linear amplification product. Accordingly, the present invention provides an isolated linear amplification product obtained using such a method. In another embodiment, the hybridization probe comprises a plurality of different hybridization probes, which can be immobilized in an array to a solid support such as a microchip, a glass slide, or a bead, such a plurality of probes being useful, for example, to obtain a hybridization pattern characteristic of a cell, which can be a normal cell, including a cell of a particular tissue or at a particular stage of development, or can be a cell involved in a pathologic condition, including a virus infected cell.

The present invention also relates to a kit, which contains one or more reagents useful for practicing a method of the invention. As such, a ldt of the invention can contain, for example, a target nucleic acid molecule; at least one primer, which can selectively hybridize to a nucleotide sequence of the target nucleic acid molecule and, if desired, can be a primer of an amplification primer pair; and an exonuclease. The target nucleic acid in the kit can be about 10 to 1000 nucleotides in length, generally about 50 to 500 nucleotides in length, and particularly about 100 to 250 nucleotides in length, and can be provided as a monomer, or as a concatemer of about 2 to 1000 monomeric target nucleic acid molecules, generally about 10 to 250 monomeric target nucleic acid molecules, and particularly about 25 to

100 monomeric target nucleic acid molecules; or can include a combination of monomeric and concatemerized target nucleic acid molecules, including monomers of varying length or concatemers containing various numbers of linked monomeric units.

A kit of the invention also can contain a polynucleotide that can integrate into the target nucleic acid molecule supplied with the kit. Such an integrating polynucleotide can be useful, for example, as a control to confirm or normalize results of an integration assay performed using the kit. Such a kit also can contain a second primer of an amplification primer pair, wherein the second primer selectively hybridizes to a nucleotide sequence of the integrating polynucleotide. In addition, the kit can contain an integrase that can mediate integration of the polynucleotide into a target nucleic acid molecule.

A kit of the invention also can contain, for example, a target nucleic acid molecule; at least one primer, which can selectively hybridize to a nucleotide sequence of the target nucleic acid molecule and, if desired, can be a primer of an amplification primer pair; and a polynucleotide that can integrate into the target nucleic acid molecule. Such a kit also can contain a second primer of an amplification primer pair, wherein the second primer selectively hybridizes to a nucleotide sequence of the integrating polynucleotide.

In addition, a kit of the invention can contain a detector oligonucleotide, which can selectively hybridize to a nucleotide sequence of a recombinant nucleic acid molecule formed upon integration of an integrating polynucleotide into the target nucleic acid molecule; or can selectively hybridize to an amplification product generated therefrom. The kit also can contain at least one primer of a second amplification primer pair, wherein the primer can selectively hybridize to a nucleotide sequence of an amplification product generated using the first amplification primer pair.

A kit of the invention also can contain an oligonucleotide primer, which can selectively hybridize to a selected strand of a nucleic acid molecule under certain conditions, but which forms a secondary structure that reduces or inhibits the ability to selectively hybridize to the selected strand under other conditions. Such a kit also can contain an amplification primer pair, wherein the primers of the amplification primer pair can selectively hybridize to a target nucleic acid molecule and provide substrates for amplification under condition in which the oligonucleotide primer forms the secondary structure.

The present invention also relates to a kit, which contains a master mix containing all of the components for performing an integration reaction in a single tube. As such, the single tube in such a kit contains an integrating polynucleotide, which can be a PIC, a pro-PIC, a salt-stripped PIC, or the like; and a target nucleic acid molecule; and contains a buffer that provides conditions sufficient for integration of the integrating polynucleotide into the target nucleic acid molecule. The master mix can be in a lyophilized form, or can be provided as a solution, which can, but need not, be in a concentrated form, for example, a 2X, 10X or other concentration, and can be frozen or maintained under refrigeration conditions.

Such a master mix can further contain an exonuclease such as λ exonuclease, which is in an amount that is sufficient to degrade unreacted target nucleic acid molecules following integration of the integrating polynucleotide into the target nucleic acid molecule, but is not sufficient to substantially degrade target nucleic acid molecules during the time an integration reaction is proceeding. In addition, the master mix can contain at least one primer that can selectively hybridize to the target nucleic acid molecule or to the integrating polynucleotide, and a DNA polymerase, such that a linear amplification reaction can be performed, and can further contain a second primer, wherein the first and second primer provide an amplification primer pair such that an amplification reaction such as PCR can be performed. The master mix also can contain the deoxyribonucleotide triphosphates required for the amplification, and can further contain a detector oligonucleotide, for example, a bilabeled oligonucleotide probe that can be used for real-time analysis of generation of an amplification product. Such a kit provides a convenient means to prepare standardized reaction conditions and, therefore, is suitable for preparing, for example, an array of reaction mixtures that can be used to screen test agents to identify an agent that modulates integration activity or to identify factors that are involved in integration.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic depiction of an embodiment of a pre-integration complex (PIC) integration assay.

In step 1, a simplified view of a single retroviral long terminal repeat (LTR), which terminates with a CA at its processed 3' end, is shown, as is the large excess of concatemeric target DNA present in the reaction. The multiple binding sites for the reverse PCR primer are shown as black boxes within each concatemeric target DNA. In step two, the strand transfer reaction is shown. Strand transfer results from retroviral integration in vitro, wherein an integration junction is formed by the covalent joining of the 3' end of the LTR, which contains the CA terminal residues, with the concatemeric target DNA. Regardless of where integration occurs in the concatemeric target DNA, a binding site for the reverse primer (black box) is present within 105 bp downstream of the integration junction. The "unreacted target DNAs" are shown contain an excess of binding sites for the reverse primer, that can interfere with the subsequent PCR reaction. Accordingly, unreacted target DNA is removed using λ exonuclease, which digests DNA in the 5'-to-3' direction. Proteins bound to the LTR protect the integration junction from λ exonuclease digestion. Following treatment with the λ exonuclease, proteinase K is used to remove the exonuclease and other proteins, then is heat inactivated.

In step 3, PCR detection is performed. In the first cycle, the reverse primer generates the strand complementary to the integration junction, thereby producing a double stranded amplicon containing a binding site for the forward primer (open box). Real-time PCR detection is performed using a TaqMan™ probe, which binds to a site in the LTR (hatched box) downstream of the forward primer site. The 5' exonuclease activity of Taq polymerase cleaves the fluorophore (F) in the probe from the quencher (Q), generating a fluorescent signal.

Figure 2 shows the number of integration events as determined by real-time fluorescence PCR. Eleven ng of 32-mer target DNA was used. Samples were analyzed without (-) or with (+) λ exonuclease digestion, and without dilution ("1") or following 10-fold ("10") dilution, prior to the detection step. The number of integration events produced by the original 10 μl of PIC-containing cytoplasmic extract was calculated by multiplying by the dilution factor. The data shown are the mean of duplicate samples and are representative of five experiments.

Figure 3 illustrates the dependence of integration reaction efficiency on the length and amount of target DNA. Integration reactions were conducted in the presence of known amounts of monomeric (SI) or 32-mer (S32) target nucleic acid molecules. Samples were treated with λ exonuclease and examined without dilution. The amount of target nucleic acid (X-axis) is based on the total number of monomeric units present in the reaction mixture. Data are the mean of duplicate samples and are representative of four experiments.

Figure 4 provides an enumeration of integration-competent PICs. PICs were serially diluted (X-axis) in the extraction solution and placed into integration reactions (11 ng target/reaction). Samples were treated with λ exonuclease and analyzed without dilution using the real-time PCR assay. Data are the mean of paired duplicate samples. A dose-response relationship between the number of PICs added and the number of integration events detected was observed. The linear trend of this dose- response data was highly significant as determined by one-way ANOVA (r = 0.9545, p < 0.0001). As an additional statistical measure, a single sample was analyzed in eight separate reactions and had a mean of 479 integration events with a co-efficient of variation of 31.45%.

Figure 5 shows the detection and quantification of inhibitors of PIC integration. PICs were preincubated at 37°C prior to the addition of target DNA. Samples were treated with λ exonuclease and diluted 10-fold prior to real-time PCR detection. Since the PIC-containing cytoplasmic extracts were unstable when incubated at 37°C for more than 20 minutes (inset), the PICs were preincubated for 10 min at room temperature with various concentrations of two known inhibitors of HIV-1 integrase, purpurin (■ — ■) or quinalizarin (A— A), then the target nucleic acid molecules were added. Samples were treated with λ exonuclease, and the number of integration events was determined in undiluted samples following the removal of the interfering pigmented integration inhibitors by ultrafiltration.

Figure 6 shows the integration events following reconstitution of salt-stripped

PICs with host cell factors. The dotted line shows the lower limit of sensitivity for the real-time PCR detection system. Salt-stripped PICs ("1") were not competent for integration, whereas the addition of a cell extract from SupTl CD4⁺ T cells resulted in integration-competence ("2").

Figures 7 A and 7B illustrate portions of integration junctions. Figure 7 A shows a portion of an integration junction formed between HIV-1 and a silk drag line target nucleic acid molecule (SEQ ID NO: 6). Sequences corresponding to the LTRTaq5 forward primer (italics; SEQ ID NO:l), LTRTaqP TaqMan™ probe (bold and underlined; SEQ ID NO :3), and SILKREV 1 a reverse primer (bold; SEQ ID NO:2) are indicated and bounded by the arrows. Nucleotides to the end of the 3' LTR of HIV-1 are shown, with the end of the LTR indicated by an arrow. An internal Sty I and 3' terminal Spe I and Bam HI restriction endonuclease sites (underlined) also are shown. Nucleotides of the "silk sequence" are indicated by a run of "N", wherein the specific nucleotide sequence will depend on the specific site of integration.

Figure 7B provides an example, of a target nucleic acid molecule that is concatemeric with respect to the binding sites only, with non-repeating sequences outside of the primer-binding sites (SEQ ID NO:7). "N₈o" refers to any arbitrary eighty nucleotide sequence. The forward and reverse primers (as indicated; SEQ ID NOS:8 and 10; SEQ ID NO:9 shows the sequence of SEQ ID NO:7 that is the bound by the reverse primer) can be used to amplify a population of these monomeric units. The nonsymmetric Ava I sites (underlined near 5' terminus) in the primer regions can be cut with Ava I, and the cleavage product can be used to create a head-to-tail concatemer (see below). An advantage of such a target nucleic acid molecule is that any differences in sequence between each unit in the concatemer can contribute to stability of the construct in plasmids amplified in bacteria. Figures 8 A and 8B illustrate the use of a junctional probe for detecting an integration junction.

Figure 8 A shows an integration junction of an HIV-1 3' LTR and a homopolymeric target nucleic acid molecule (underlined; SEQ ID NO: 11). "(T)" indicates any number of thymidine residues, as defined herein. The junctional probe ("Junctional Oligonucleotide"; SEQ ID NO: 12) is shown spanning the integration junction.

Figure 8B shows an integration junction of an HIV-1 3' LTR and a target nucleic acid molecule containing CT repeat units (underlined; "alternating nucleotide target nucleic acid molecule"). "(TC)" indicates any number of thymidine and cytidine residues, as defined herein. The upper panel illustrates integration of the HIV LTR adjacent to a T residue in the target nucleic acid molecule (SEQ ID NO: 13) and the lower panel illustrates integration of the HIV LTR adjacent to a C residue in the target nucleic acid molecule (SEQ ID NO: 15). Junctional probes that selectively hybridize to the integration junction shown in the upper panel and in the lower panel are indicated as "Junctional Oligo-1" (SEQ ID NO: 14) and "Junctional Oligo-2" (SEQ ID NO: 16), respectively, and span the respective integration junctions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for detecting the formation of a covalent linkage formed between two nucleic acid molecules, including for detecting integration of a polynucleotide into a target nucleic acid molecule. The methods of the invention can be conveniently performed using readily available reagents, and are adaptable to a high throughput format. Accordingly, the methods of the invention provide a means to identify agents that can modulate the ability of a polynucleotide to integrate into a target nucleic acid molecule, and to identify factors involved in such integrating events.

A method of the invention can be performed, for example, by contacting a sample, including an integrating polynucleotide and a target nucleic acid molecule, under conditions that allow integration of a polynucleotide into the target nucleic acid molecule; and detecting covalent linkage of the integrating polynucleotide and the target nucleic acid molecule. The term "integrating polynucleotide" is used broadly herein to refer to a nucleotide sequence that can be enzymatically inserted into a second nucleotide sequence, wherein the enzymatic activity includes a cleavage activity and a ligase activity. Integrating polynucleotides are exemplified by nucleotide sequences that are inserted into to a second nucleotide sequence due to the action of an integrase, a transposase, a recombinase, or the like. Such integrating polynucleotides include, for example, viral polynucleotide such as a nucleotide sequence of a retrovirus, including a retrovirus of a fish, amphibian, reptile, bird, or mammal, for example, a retrovirus genome such as a human immunodeficiency virus (HIV) genome such as an HIV-1 or HIV-2 viral genome, as well as a polynucleotide portion of such a retrovirus genome, for example, a long terminal repeat (LTR); a transposable element such as a yeast transposon, a plant transposon, and a Drosophila P element, including at least the nucleotide sequence of one or both termini having integration activity; a retroelement such as occurs in a eukaryotic genome, including in a vertebrate genome; nucleotide sequences of mammalian genes such as immunoglobulin superfamily genes, which undergo rearrangements, and other nucleotide sequences generally known in the art as transposable or mobile genetic elements.

Reference herein to "conditions that allow integration of a polynucleotide into the target nucleic acid molecule" means that a sample in which the integration reaction is being performed are appropriate for integration to occur. Such conditions are exemplified in Example 1 and include, for example, appropriate buffer capacity and pH, salt concentration, metal ion concentration if necessary for the particular integrase, transposase, or the like. In addition, such conditions include the presence of the appropriate enzyme that mediates the integration event, as well as necessary viral or cellular factors (depending on the particular integrating polynucleotide). As disclosed herein, an aspect of the invention provides a means to identify and, if desired, isolate the enzymes or viral or cellular factors involved in an integration event, and, similarly, provides a means to identify and, if desired, isolate a polynucleotide having the characteristics of an integrating polynucleotide, including a synthetic, recombinant, or naturally occurring polynucleotide.

Similarly, reference herein to "conditions that allow generation of an amplification product" or of "conditions that allow generation of a linear amplification product" means that a sample in which the amplification reaction is being performed contains the necessary components for the amplification reaction to occur. Examples of such conditions are provided in Example 1 and include, for example, appropriate buffer capacity and pH, salt concentration, metal ion concentration if necessary for the particular polymerase, appropriate temperatures that allow for selective hybridization of the primer or primer pair to the template nucleic acid molecule, as well as appropriate cycling of temperatures that permit polymerase activity and melting of a primer or primer extension or amplification product from the template or, where relevant, from forming a secondary structure such as a stem-loop structure. Such conditions and methods for selecting such conditions are routine and well known in the art (see, for example, Innis et al, "PCR Strategies" (Academic Press 1995); Ausubel et al, "Short Protocols in Molecular Biology" 4th Edition (John Wiley and Sons, 1999)).

As used herein, the term "selective hybridization" or "selectively hybridize" refers to hybridization under moderately stringent or highly stringent conditions such that a nucleotide sequence associates with a selected nucleotide sequence but not with unrelated nucleotide sequences. Generally, an oligonucleotide useful as a probe or primer that selectively hybridizes to a selected nucleotide sequence is at least about 15 nucleotides in length, usually at least about 18 nucleotides, and particularly about 21 nucleotides in length or more in length. Conditions that allow for selective hybridization can be determined empirically, or can be estimated based, for example, on the relative GC:AT content of the hybridizing oligonucleotide and the sequence to which it is to hybridize, the length of the hybridizing oligonucleotide, and the number, if any, of mismatches between the oligonucleotide and sequence to which it is to hybridize (see, for example, Sambrook et al., "Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989)). Numerous functions are performed in diverse organisms by integrating polynucleotides. For example, ciliates use recombinases to radically process the DNA of the germline micronucleus as the somatic macronucleus is created. Nematodes use a similar programmed expression of transposases to convert the germline chromosomes to radically different somatic chromosomes (Goday and Pimpinelli 1993). Drosophila uses two non-LTR retrotransposons (HeT-A and TART) to maintain telomeres (Pardue et al. 1997). In zebrafish, the rag 1 recombinase is expressed in the olfactory epithelium as well as in tissues in which common and variable genes are switched in the immune system (Jessen et al. 1999; for additional examples mobile genetic elements and their functions in organisms, see, for example, Patrusky 1981; Bostock 1984; Williams et al. 1993; Medstrand and Blomberg 1993; Goto et al. 1998; Plasterk, Trends Genet. 1992).

The mechanism for excision of genomic DNA during the development of the immune system utilizes mechanisms and enzymes that evolved with mobile elements such as DNA transposable elements and retroelements (Spanopoulou et al. 1996; Landy 1999). At least ten to twenty percent of the genomic DNA of most multicellular organisms is composed of nucleotide sequences that are related to mobile genetic elements, and a large numbers of genes coding for members of the transposase/recombinase family are present in these genomes. For example, during heavy chain switching in the immune system via reverse transcriptases and the related nucleases, an RNA transcript functions in a manner that is similar to the processes used by retroelements (Muller et al. 1998). Thus, a site-specific nuclease appears to nick genomic DNA in a region of repeats (splice region), the RNA forms a heterodimer with the DNA in the nicked region, and a reverse transcriptase copies the RNA. The result is excision of a circular DNA molecule and the joining of the edited DNA to form a new protein coding sequence (exons and introns), control regions, and the like. B cell specific retroelements are expressed in these cells and may be the source of the required reverse transcriptase and nuclease activities. Retroelements are mobile genetic elements that can exist in the form of DNA, RNA or DNA/RNA duplexes. Although retroviruses are well known retroelements, there are many other types, including close relatives of retroviruses such as LTR retrotransposons, more distant relatives like non-LTR retrotransposons, caulimoviruses and hepadnaviruses, and elements with virtually no similarity such as retrons. Except for telomeres and telomerases, which maintain the ends of chromosomes, most retroelements have been considered to be "selfish DNA" that is not involved with the normal development or maintenance of their host cells, (Flavell, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 110:13-15, 1995).

Retroviruses are one of the best known examples of integrating polynucleotides. Besides their role in causing disease, retroviruses have been extensively studied as vectors for gene therapy. Retroviruses include oncoviruses such as avian leukemia virus (ALV), a Rous sarcoma virus (RSN), Mason-Pfizer monkey virus, and simian retrovirus type 1 and type 2; lentiviruses such as human immunodeficiency virus (HIN) type I (HIN-I) and type II (HIV-II); and spumaviruses. Retroviruses have a two stage life cycle, existing in an RΝA form and a DΝA form. The RΝA form of the virus is packaged into an infectious particle that is coated with a glycoprotein (env), which is recognized by receptors on the host cell. This interaction promotes a receptor mediated internalization event, resulting in exceptionally efficient delivery of the retroviral genome into the cell.

After entering the cell, the RΝA genome is reversed transcribed in the cytoplasm by RΝA-dependent DΝA polymerase (reverse transcriptase) associated with the infecting viral vector. A pre-integration complex (PIC) harboring the reverse transcribed viral genome then enters the nucleoplasm. The size of the PIC appears to preclude passive entry into the nucleus; in the case of C type retroviruses the infected cell must be dividing for the complex to reach the host cell genome. While retroviruses do not integrate in a site-specific manner, they also do not integrate in a completely random fashion, but generally have a preference for integration into regions of the genome that are transcriptionally competent. This characteristic reduces the likelihood that the transcription of coding sequences of the pro virus will be silenced by integration into a transcriptionally repressive domain.

In a recombinant retroviral vector, coding sequences of the virus generally are replaced with a nucleotide sequence of interest, for example, a transgene encoding a polypeptide of interest. This replacement is done by standard molecular biological techniques using a pro viral version of the virus that is propagated as a bacterial plasmid (a retroviral vector plasmid). Certain sequences of the retrovirus are maintained, including, for example, all or most of the components of the LTR, the packaging signal, and the primer binding site. However, other sequences of the retrovirus genome, including, for example, the genes encoding the gag, pol and env proteins, which are absent in the retroviral vector genome, are required for transcription and packaging of a viral particle. While such sequences can be removed from the retroviral vector genome without substantially inhibiting the functional activity of the vector genome, in order to obtain a fully functional recombinant retroviral vector, the proteins encoded by these sequences must be provided in trans, for example, by expression from other plasmids that are introduced into the host cell via cellular transfection. Alternatively, these helper functions can be designed to already be integrated into the cellular genome of the viral packaging line (helper cell).

Retroviruses have two viral promoters (LTRs), one located at each end of the proviral form of the retroviral genome. The upstream LTR is responsible for promoting transcription of the DNA provirus into the RNA form. Transcription starts at the 5' border of the R region in the upstream LTR and terminates within the R region of the downstream LTR. As such, the RNA form of the retrovirus contains a U5 region copied from the upstream LTR and a U3 region copied from the downstream LTR. During reverse transcription of the RNA into the DNA provirus, both upstream and downstream LTRs are formed from the R region and sequences comprising the U5 and U3 regions found at the 5' and 3' ends respectively of the RNA genome. Thus, native, wild-type retroviruses contain identical sequences in their upstream and downstream LTRs. A retroviral vector plasmid generally contains a retrovirus LTR, including a U5 region, an R region and a U3 region; and a cloning site.

An integrating polynucleotide useful in a method of the invention can be any polynucleotide that can integrate into a target nucleic acid molecule. As such, the integrating polynucleotide can comprise a retrovirus LTR or an integrating portion thereof; or can comprise a nucleotide sequence generally found at one or both ends of a transposon or other mobile genetic element. Although only the minimal sequence required for integration is required, additional nucleotide sequences can be included in the integrating polynucleotide, including, for example, coding sequences, regulatory elements, and the like. For example, the integrating polynucleotide can be a retroviral vector constructed for a gene therapy procedure, or a portion thereof, which is being examined for an ability to integrate in the presence of a drug that is being considered to be coadministered with the vector to a patient. The integrating polynucleotide also can be a putative integrating polynucleotide, for example, a nucleotide sequence obtained from genomic DNA of an organism, including a mammalian organism such as a human, that can, but need not, have characteristics of a retrovirus or other retroelement, wherein a method of the invention is utilized to determine whether the putative integrating polynucleotide, in fact, has integrating activity.

An integrating polynucleotide generally requires an integrase, transposase, recombinase, or other similar enzyme or combination of enzymes to exhibit integrating activity. Accordingly, such enzymes and other requisite factors are included with the polynucleotide in a sample containing the target nucleic acid molecules. Such additional factors can be added as relatively purified components, for example, an isolated integrase or transposase, or can be included within an extract such as a cell extract known to contain the factors. For example, where the integrating polynucleotide is a viral polynucleotide, the polynucleotide can be added to a sample and an extract prepared from cells infected with the virus also can be added, thus providing the components required for integration. Alternatively, a preintegration complex (PIC), which contains the viral polynucleotide in a complex with the necessary factors for integration can be obtained from a virally infected cell, and the PIC can be added to the sample containing the target nucleic acid molecule.

Retroviral PICs generally are obtained by detergent extraction of host cells infected with a retrovirus such as HIV. As a result, the PICs lack the viral envelope and other proteins of the original infectious virus. Nevertheless, the procedure generally is performed under biosafety containment conditions, as there is a theoretical concern in using such PICs on the open bench. Such a risk can preclude use of the potentially infectious PICs in an industrial process such as high-throughput screening. Accordingly, a method of the invention also can be performed using non-infectious PICs.

Non-infectious PICs can be prepared as previously described (Hansen et al., Nat. Biotechnol. 17:578-82, 1999; U.S. Pat. No. 6,218,181, each of which is incorporated herein by reference) using, for example, a replication-defective lentiviral vector (see, for example, Naldini et al, Science 272:263-267, 1996). The replication- incompetent lentiviral genome can be produced in 293T cells, which are engineered to express the envelope of vesiculostomatitis virus (VSV-G protein). The resulting pseudotyped virions then are used to infect the cells of interest and PICs are harvested from those infected cells. Cell-to-cell spread of virus does not occur in these cultures because the virus is genetically defective, and the system does not utilize the natural replicative ability of retroviruses.

Replication-deficient HIV-1 clone RS4X, which is defective in the rev gene, was cultured in a helper cell line, which complements the genetic defect in the virus. The rev gene product, which is required for HIV-1 replication, was provided by culturing the defective virus in CEMrevl helper cells, which are lymphoblastoid cells that have been engineered to stably express HIV-1 rev (Riggs and Guatelli, Virology 217:602-6, 1996). The HIV-1 virions that were produced spread through the CEMrevl culture, and the supernatant of this culture was used as the viral stock. The viral stock was used to infect CEM cells or SupTl cells, creating PICs within these cells. The PICs were integration competent using the concatemeric S32 silk target DNA. In a control experiment, HIV-1 clone RS4X was introduced into human cells that had not been modified to express HIV-1 rev. The infected cells were cultured for up to one week and examined for the production of viral capsid p24 antigen. No p24 antigen was detected in the supernatant, confirming that the virus was unable to replicate in cells that have not been engineered to express HIV-1 rev and, therefore, that the PICs are non-infectious in human cells.

A similar method uses specialized virus and specialized host cells to produce virus that is not infectious to normal cells. In this case, the virus can be entirely intact except for a deletion introduced into the env gene. As an example, the proviral HIV-1 clone, pBruDeltaEnv, can be transfected into 293T cells where all of the viral proteins except for the envelope protein are produced (Bartz et al., Methods 12:337-342, 1995. If the same producer 293 T cells are also co-transfected with the modified envelope of avian leukosis-sarcoma virus subgroup A (ALSV-A), then the resulting virions are pseudotyped with the envelope of this chicken retrovirus. These virions can infect mammalian host cells engineered to express a chicken cell receptor (TV A) that is not normally expressed in mammalian cells. As a result, the virions can infect the mammalian TVA-expressing cells, but are non-infectious for other mammalian cells (Lewis et al., J. Virol. 75:9339-9344, 2001), thus providing an additional safety barrier to human infection.

The cDNA in retroviral PICs also can be selectively degraded without affecting the ability of the LTR ends to integrate into a target nucleic acid molecule in vitro. In an integration assay of the invention, in which the polynucleotide comprises a PIC, only integration of the ends of the cDNA is measured. Thus, methods can be utilized to selectively damage the intervening portions of the viral cDNA so that the genome is disrupted. For example, the cDNA can be cleaved using a restriction endonuclease or ribozyme that is selective for retroviral cDNA sequences not present in the termini (Raillard and Joyce, Biochemistry 35:11693-11701, 1996), or sequence-specific chemical nucleases such as a nucleic acid molecule or protein that can bind a specific sequence of the viral cDNA coupled to a 1,10-phenanthroline- copper complex (Francois et al., Proc. Natl. Acad. Sci.. USA 86:9702-9706, 1989; Pendergrast et al., Science 265:959-62, 1994; Sigman et al., Nature 363:474-475, 1993; Pan et al., Mol. Microbiol. 12:335-342, 1994; Perrin et al, Prog. Nucl. Acid Res. Mol. Biol. 52:123-151, 1996).

Another alternative provides for the treatment of PICs with a high concentration of salt, thereby producing salt-stripped PICs, which contain the integrating polynucleotide associated with some, but not all, of the factors required for integration. Such salt-stripped PICs can be added to a sample containing target nucleic acid molecules, and additional cellular or viral factors can be added, thus providing a means to identify the factor or factors involved in integration. Such additional factors can be isolated in fractions from virus infected cells, or from cells that have been stimulated with chemicals such as cyto ines or other molecules that can simulate a virus infection. Similarly, integrating polynucleotides comprising all or a portion of a transposon or other mobile genetic element, and the factors required for integration can be obtained using the appropriate starting materials, particularly cells known to contain the polynucleotides.

As mentioned above, upon entering a cell and undergoing reverse transcription, the retroviral genome is contained in a PIC, which mediates integration of the retroviral genome into the host cell DNA. The determination that integration of HIV into a host cell genome requires the integrase enzyme (Hansen et al., Genet. Eng. (NY) 20:41-61, 1998; Asante-Appiah and Skalka, Adv. Virus Res. 52:351-369, 1999), and that mutations that destroy integrase activity block viral replication (Wiskerchen and Muesing, J. Virol. 69:376-386, 1995), established integration as an important target for the development of antiretroviral drugs (Moore and Stevenson, Nat. Rev. Mol. Cell Biol. 1 :40-49, 2000). As such, attempts have been made to develop in vitro assays that measure retrovirus integration and that can be used to screen for agents that affect integration. One such assay, a modified strand transfer assay, in which a pre-assembled complex was formed between recombinant integrase proteins and oligonucleotides designed to model the ends of the viral cD A, was used to screen a library of 250,000 compounds, and identified a class of integrase inhibitors, including several di-keto compounds that inhibited HIV infection in CD4+ T cells in vitro (Hazuda et al., Science 287:646-650, 2000; Hazuda et al., Nucl. Acids Res. 22: 1120- 1122, 1994; Craigie et al, Nucl. Acids Res. 19:2729-2734. 1991).

Although the results obtained using the strand transfer assay indicated that integration inhibitors could become a new treatment modality, the assay scored as positive occasional compounds that failed to inhibit integration in vitro by authentic PICs isolated from the cytoplasm of infected cells or virus-mediated integration in cultured cells (Hazuda et al., supra, 2000; Farnet et al. Proc. Natl. Acad. Sci.. USA 93:9742-9747, 1996). This result indicates that other components of the PIC, which includes viral proteins such as nucleocapsid and host cell factors (see, for example, Bukrinsky et al, Proc. Natl. Acad. Sci.. USA 90:6125-6129, 1993; Lee and Craigie, Proc. Natl. Acad. Sci.. USA 91:9823-9827, 1994; Miller et al., J. Virol. 71:5382-5390, 1997; Chen and Engelman, Proc. Natl. Acad. Sci.. USA 95:15270-15274, 1998) are essential for the efficient integration of retroviral cDNA.

The host cellular factors involved in integration mediated by PICs can provide additional targets for drugs that modulate integration. However, relatively little is known about PIC-mediated integration as an enzymatic process, and it has been difficult to construct high throughput 'assays for examining integration mediated by PICs. An examination of the influence of the target nucleic acid molecule on PIC integration revealed that PICs preferentially integrate into regions of distorted DNA such as occurs in nucleosomes (Bor et al., Proc. Natl. Acad. Sci.., USA 92:10334- 10338, 1995; Pryciak and Varmus Cell 69:769-780, 1992; Pruss et al, Proc. Natl. Acad. Sci., USA 91:5913-5917, 1994) and tend to avoid sequences upstream of a pyrimidine nucleotide (Bor et al, Virology 222:283-288, 1996).

PICs preferentially bind torsionally strained DNA. However, the effect of the length of a target nucleic acid molecule, which can affect substrate mobility, on retroviral integration has not been reported. As disclosed herein, concatemerization of a repeating 105 base pair unit was used to vary target DNA length independently of the target nucleic acid sequence. PICs and target nucleic acid molecules were maintained in solution, and integration junctions were quantified by real-time fluorescence-monitored PCR amplification using primers specific for the viral LTR and the target nucleic acid molecule. Unreacted target nucleic acid molecules were found to inhibit detection of generated amplification product and, therefore, were removed by digestion with λ exonuclease or were reduced in concentration by dilution. Integration into a 32 unit concatemer target nucleic acid molecule was markedly more efficient than integration into a monomeric unit at the same molar concentration (Example 1). These results demonstrate that a relatively longer target nucleic acid molecule is a preferred substrate for retroviral integration, and provide the basis for a simple and robust integration assay that is conveniently adaptable to a high throughput format and can serve as a drug discovery platform to identify agents that can modulate such integration.

Accordingly, the present invention provides methods of detecting integration of a polynucleotide into a target nucleic acid, including methods of detecting covalent linkage of the polynucleotide to the target nucleic acid molecule. As used herein, the term "target nucleic acid molecule" or "target molecule" means a nucleotide sequence to which an integrating polynucleotide can be covalently linked. A target nucleic acid molecule can be double stranded or single stranded DNA or RNA, and is characterized, in part, in having a nucleotide sequence to which an oligonucleotide primer can specifically hybridize such that an extension product spanning the integration junction can be generated.

Since an integrating polynucleotide that generally is examined according to a method of the invention does not undergo site-specific integration, a target nucleic acid molecule can have any sequence, including, for example, a coding sequence, intronic sequence, or other nucleotide sequence such as a regulatory element generally found upstream or downstream of a coding sequence in a genome; a repetitive nucleotide sequence such as a moderately repetitive or highly repetitive sequence, as is generally present in a mammalian or other vertebrate or eukaryotic genome; a nucleotide sequence containing a repeat of a single nucleotide such as a polyadenosine sequence, oligothymidine sequence, polyuridine sequence, or the like; a randomly generated nucleotide sequence containing two or more different nucleotides; or any other nucleotide sequence. A target nucleic acid molecule generally is about 10 to 1000 nucleotides in length, usually about 50 to 500 nucleotides in length, and particularly about 100 to 250 nucleotides in length, such that the target nucleic acid molecule is an effective substrate for integration and, further, allow an integration event to be detected by a method such as an amplification reaction, for example, a polymerase chain reaction (PCR). As such, the target nucleic acid molecule is constructed such that, regardless of the position of the integration event, an extension product generated using a primer, which selectively hybridizes to a particular nucleotide sequence of the target nucleic acid molecule, includes the integration junction, i.e., at least a part of the target nucleic acid molecule sequence and a part of the integrating polynucleotide sequence (see Figure 1).

A target nucleic acid molecule can be a monomeric target molecule, or can be constructed as a concatemer of monomeric target nucleic acid molecules. A concatemerized target nucleic acid molecule can contain from about 2 to 1000 linked monomeric target nucleic acid molecules; generally contains about 10 to 250 linked monomeric target nucleic acid molecules, and particularly contains about 25 to 100 linked monomeric target nucleic acid molecules. Generally, each monomeric target nucleic acid molecule contains a nucleotide sequence that can be selectively hybridized by an oligonucleotide primer, which can act as a substrate for an extension reaction such as a primer extension or linear amplification, or can be one of an amplification primer pair. As disclosed herein, a concatemerized target nucleic acid molecule provides a particularly effective substrate for integration, and further provides a target in which integration events conveniently can be detected by an amplification reaction such as PCR.

A concatemeric target nucleic acid molecule can be prepared using well known and routine methods. For example, standard recombinant DNA methods can be used to ligate two or more nucleotide sequences together into a plasmid. The insert then can be excised and used as a concatemerized target nucleic acid molecule, or two or more of the excised inserts can be ligated together into another plasmid, generating, for example, four monomeric units. Such a process can be performed as many times as desired, and the number of monomers in the concatemers can be determined, for example, by agarose gel electrophoresis (see,- for example, Hardies et al., J. Biol. Chem. 254:5527-34, 1979; Sadler et al, Gene 8279-300, 1980; Shen, Proc. Natl. Acad. Sci.. USA 81:4627-31, 1984; Kempe et al., Gene 39:239-45, 1985, each of which is incorporated herein by reference). Such a method was used to produce the exemplified concatemeric target nucleic acid molecule encoding drag-line silk (Prince et al., Biochemistry 34:10879-85, 1995) and has been used to produce concatemeric nucleic acid molecules encoding other proteins having a repeating structure (U.S. Pat. No. 5,641,648, each of which is incorporated herein by reference, which is incorporated herein by reference) .

A concatemerized target nucleic acid molecule also can be generated using a nonsymmetrical Ava I restriction endonuclease site, which is incorporated at each end of the monomeric units to be concatemerized (Figure 7B; see, also, Hartley and Gregory, Gene 13:347-53, 1981). Upon cleaving the sequences with Ava I, the ends that are generated can only be ligated in a head-to-tail manner. The ligation product then can be cloned into a vector, which can also contain the same nonsymmetrical Ava I site and can serve as an "initiator" for a ligase-mediated polymerization reaction (Graham and Maio, BioTechniques 13:780-789, 1992). A further modification, in which the cloning plasmid is digested into two halves, each of which is then used as the initiator in a separate ligase-mediated polymerization reaction, also can be used to generate the concatemeric target nucleic acid molecule (Radlinska et al., BioTechniques 31:340-347. 2001).

A concatemeric target nucleic acid molecule also can be prepared by cloning using T7 bacteriophage, which naturally forms a concatemeric DNA (U.S. Pat. Nos. 5,030,566 and 5,294,545). A concatemer chain reaction method also can be utilized, wherein a pair of partially complementary oligonucleotides, which contain unpaired ends that hybridize to their opposite strand, are extended with respect to each other in the presence of DNA polymerase, particularly a thermostable DNA polymerase, and dNTPs are added (Doel et al. Nucl. Acids Res. 8:4575-4592, 1980; Rudert and Trucco, Nucl. Acids Res. 18:6460, 1990; White et al, Anal. Biochem. 199:184-190, 1991; Ijdo et al., Nucl. Acids Res. 19:4780, 1991).

A concatemeric target nucleic acid molecule also can be prepared using the "rolling circle" method, which utilizes a circular DNA template, a primer that binds to this circular DNA, DNA polymerase, and dNTPs (Fire and Xu, Proc. Natl. Acad. Sci.. USA 92:4641-4645, 1995; Lizardi et al, Nat. Genet. 19:225-32, 1998; U.S. Pat. Nos. 5,648,245 and 5,714,320). The rolling circle method can be performed, for example, using a DNA polymerase that has high strand-displacement activity, because the polymerase extends the primer completely around the circular D A template and then has to advance beyond the primer binding site by displacing the hybridized primer. Examples of DNA polymerases with strong strand displacement activity include Phi29 DNA polymerase (TempliPhi, Amersham-Pharmacia Biotechnologies) and Bst I DNA polymerase (Hafner et al., BioTechniques 30:852-856, 2001). Where rolling circle DNA replication generates a single stranded concatemer, a complementary inverse primer can be used to generate the complementary strand, if desired.

As disclosed herein, an assay, which can be performed in solution in a fluid phase, has been developed for detecting integration junctions in vitro. As used herein, the term "integration junction" refers to the site of a covalent bond formed between an integrating polynucleotide and a target nucleic acid molecule, and can include nucleotide sequences of the integrating polynucleotide, the target nucleic acid molecule, or both. Only integration junctions that form due to covalent linkage of the integrating polynucleotide, for example, a viral cDNA, and a target nucleic acid molecule are scored as positive using the method of the invention. In one aspect, the method utilizes an amplification reaction to detect integration, and a target nucleic acid molecule substrate that allows the generation of an amplification product, which is indicative of an integration event, within the optimum limits for real-time fluorescence-monitored PCR detection. In another aspect, the amplification reaction is a linear amplification reaction. By comparing a single 105 base pair (bp) target nucleic acid molecule with concatemers of the monomeric sequence, a preference of PICs for longer target nucleic acid molecules was identified. Using an integration assay of the invention, host cell factors that reconstituted the integration-competence of salt-stripped PICs also were identified (see Example 1).

PCR amplification is a highly sensitive and time-efficient method for detecting the covalent joining of two DNA molecules. In addition, a highly sensitive real-time fluorescence-monitored PCR method provides a way to obtain accurate quantification of a PCR product within a two to three hour period. However, the real-time PCR method has maximum amplification efficiency and sensitivity for small amplicons, whereas host cell genomic DNA that is a natural target of retroviral integration has substantial length, and structures that are characteristic for longer DNA molecules. To mimic this long target DNA, plasmid DNA has been employed as a target DNA in conjunction with PCR-based detection using LTR-specific and plasmid DNA-specific oligonucleotides (Shibagaki and Chow, J. Biol. Chem. 272:8361-8369, 1997). However, the length of the PCR product amplified as a result of integration varied significantly because the retroviral genome integrated nearly randomly into the target DNA and, therefore, the assay utilizing plasmid DNA as a target nucleic acid molecule could not be adapted for real-time PCR detection of integration events. The use of a short nucleic acid molecule as a target can be used in an integration assay, thus placing an upper bound on the length of an amplicon containing the integration junction. However, a short target nucleic acid molecule bears little resemblance to host genomic DNA, and is not an ideal substrate for integration (see Figure 3).

As disclosed herein, the use of a relatively long concatemeric nucleic acid molecule as an integration target overcomes the limitations discussed above. An integration assay of the invention is exemplified using PICs isolated from HIV-1 infected cells. By designing a "forward" amplification primer that selectively hybridizes at or near the end of the HIV 3' LTR and a "reverse" primer that selectively hybridizes to a nucleotide sequence present in each 105 bp monomeric unit of the exemplified concatemerized target nucleic acid molecule, amplicon size was maintained within limits optimal for efficient detection, for example, for efficient real-time PCR detection. Several thousand integration events were routinely detected using ten microliters of PIC extract prepared from virus infected cells (Example 1).

The use of a concatemeric target nucleic acid molecule provided an opportunity to conduct a systematic study of the relationship between the length of the target and the efficiency of HIV-1 cDNA integration. This analysis was performed under conditions where the sequence of the target remained constant, and only the degree of multimerization varied. When the results of this analysis was normalized in terms of an absolute number of monomeric units present in an integration reaction, concatemerization of the target nucleic acid molecule resulted in about a two-fold to ten-fold increase in the number of integration events detected, which also depended on the concentration of target molecules (number of monomers) present in the integration reaction (Figure 3).

Large amounts of double stranded DNA can suppress PCR (Kainz et al.,

BioTechniques 28:278-282, 2000; Kainz, Biochim. Biophys. Acta 1494:23-27, 2000). In the present study, the excess target nucleic acid molecule for the integration reaction that was carried over to the PCR detection step appeared to be a major inhibitor of PCR amplification, and may be the reason that a quantitative PIC integration assay using PCR to detect integration junctions has not previously been described. As disclosed herein, the inhibition of PCR detection due to unreacted target nucleic acid molecules was substantially reduced by dilution of the integration reaction mixture prior to detection; or by selective degradation of target DNA by λ exonuclease. Furthermore, the combination of λ exonuclease treatment and 10-fold dilution resulted in the highest number of integration events detected in a given sample, after adjustment was made for the dilution (Example 1).

The ability of PICs to integrate decays within about one hour at 37°C under the conditions studied (see Figure 5, inset). This decay in integration events can be due to autointegration by PICs resulting from intramolecular or intermolecular interactions with HIV-1 cDNA (Farnet and Haseltine, J. Virol. 65:6942-6952, 1991). In this context, the preference for a longer target nucleic acid molecule can reflect an improvement in "target commitment", which can be caused by decreased diffusion of the target nucleic acid molecule away from the PIC. Target nucleic acid molecule competition studies established that cytoplasmic extracts contain factors that interfere with the ability of PICs to quickly integrate into a target molecule that is contacted (Miller et al, Curr. Biol. 5:1047-1056, 1995). The decreased diffusion of a long target DNA molecule can prolong the period of contact with a PIC, thereby facilitating the proposed target-induced conformational changes in the arrangement of integrase molecules on the LTR ends (Gao et al., EMBO J. 20:3565-3576, 2001), before the decay of PIC activity becomes a limiting factor.

Using the disclosed nucleic acid integration assay, the number of integration- competent PICs in a sample were quantified over a dynamic range of about 1 to 1.5 orders of magnitude, depending on the amount of target nucleic acid molecules used. Integration events from 10 μl samples containing as few as 5 x 10⁴ copies of cDNA could be measured with a high degree of reproducibihty. The reliable measurement of integration events is particularly important for the development of integration inhibitors and for studies on the mechanism of retroviral integration. Using the disclosed methods, the activity of two previously described integrase inhibitors, purpurin and quinalizarin, was demonstrated to have IC₅₀ values similar to published values (Farnet et al, supra, 1996). In addition, reconstitution of salt-stripped PICs by cytoplasmic extracts from uninfected host cells demonstrated that the present methods can be used to detect inhibitors of integration that target a factor other than the viral integrase or other viral protein and, therefore, can be used as antiviral therapeutic agents. Furthermore, the integration assay requires minimal sample manipulation, can be performed in a single tube, and can provide quantitative data within a few hours.

The present invention provides a method of detecting integration of a polynucleotide and a target nucleic acid molecule. In one embodiment, such integration is detected by detecting covalent linkage of an integrating polynucleotide to the target nucleic acid molecule. Such covalent linkage can be detected in any of various ways, including, for example, by contacting the sample with an exonuclease having 5' exonuclease activity, which degrades unreacted target nucleic acid molecules, then contacting the sample with at least one primer that selectively hybridizes at or near the 3' terminus of the target nucleic acid molecule, under conditions that allow primer extension from the oligonucleotide primer; and detecting generation of the primer extension product. In one embodiment, the primer extension reaction is a linear amplification reaction, wherein the primer extension reaction is performed over a number of cycles, and the linear amplification product that is generated is detected.

A primer extension or linear amplification product can be detected, for example, by further contacting the sample with an amplification primer pair, which includes a first primer that selectively hybridizes to a nucleotide sequence of the integrating polynucleotide, and a second primer that selectively hybridizes to a nucleotide sequence of the primer extension or linear amplification product, under conditions that allow generation of an amplification product from the amplification primer pair molecule; and detecting generation of the amplification product. The conditions can, but need not, be such that they provide a melting temperature below that which allows selective hybridization of the primers of the amplification primer pair, and above that which allows selective hybridization of the oligonucleotide primer used for the linear amplification reaction. In one embodiment, the oligonucleotide primer is designed such that it can form a secondary structure, which can prevent the oligonucleotide primer from selectively hybridizing at or near the 3' terminus of the target nucleic acid molecule, and the conditions that allow generation of the amplification product comprise conditions below the melting temperature of the secondary structure, thereby preventing selective hybridization of the oligonucleotide primer to the target nucleic acid molecule.

Detecting covalent linkage of an integrating polynucleotide and the target nucleic acid molecule also can be performed by contacting the sample with a first amplification primer pair, which includes a first primer that selectively hybridizes to a nucleotide sequence of the integrating polynucleotide, and a second primer that selectively hybridizes to a nucleotide sequence of the target nucleic acid molecule, under conditions that allow generation of a first amplification product from the primer pair; and detecting generation of an amplification product. The amplification product that is detected can be the first amplification product, which can be detected directly, for example, by gel electrophoresis, capillary gel electrophoresis, or the like; or can be detected indirectly, for example, by further contacting the sample with a detector oligonucleotide, which can selectively hybridize to a nucleotide sequence of the first amplification product; and detecting selective hybridization of the detector oligonucleotide to the first amplification product; or by further contacting the sample with a bilabeled oligonucleotide probe such as a molecular beacon or TaqMan™ probe, which includes a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the first amplification product; and detecting fluorescence due to the fluorescent moiety. Such a method provides a means for real-time detection of the generation of an amplification product. It should be recognized that a molecular beacon, wherein the fluorescence is generated due to hybridization of the probe, which displaces the quencher moiety from proximity of the fluorescent moiety due to disruption of a stem-loop structure of the bilabeled oligonucleotide, or a TaqMan™ probe, wherein the fluorescence is generated due to degradation of the probe, which displaces the quencher moiety from the fluorescent moiety, also can be included during a linear amplification reaction and, therefore, can be used to detect generation of the linear amplification product.

The amplification product that is detected according to a method of the invention also can be a second amplification product, in which case the method further includes contacting the sample with at least a first primer of a second amplification primer pair, which can selectively hybridize to a nucleotide sequence of the first amplification product, under conditions that, in the presence of a second primer of the second amplification primer pair, allows the generation of a second amplification product. With respect to the second amplification primer pair, the second primer can be one of the first primer or second primer of the first amplification primer pair, or can be a primer that is newly added to the sample. It should be recognized that reference herein to a "first primer" or a "second primer" of an amplification primer is made for convenience only, and is not intended to indicate any importance, order of addition, or the like. It will be further recognized that an amplification primer pair requires that the first and second primer comprise what are commonly referred to as a forward primer and a reverse primer, which are selected using well known and routine methods such that an amplification product can be generated therefrom.

Real-time detection of an amplification reaction is not particularly adaptable to a high throughput format, as it requires commitment of a single expensive machine for about three hours to process a single 96 well plate. In comparison, an endpoint detection method can be readily adaptable to high throughput assays. For example, an amplification reaction such as PCR can be performed using inexpensive robotic thermocyclers for a specified number of cycles, then the amplification product generated can be determined at the endpoint of the reaction. Various endpoint detection formats are lαiown to the art and can be applied to the present methods. For example, PCR can be performed using TaqMan™ reagents, followed by reading the plates at this endpoint. Molecular beacons, Amplifluor™ or TriStar™ reagents and methods similarly can be used (Stratagene; Intergen). If the target nucleic acid molecules are sufficiently degraded with an exonuclease, SybrGreen also can be used.

PCR products also can be measured using an ELISA format, for example, using a design in which one primer is biotinylated and the other contains digoxygenin. The amplification products are then bound to a streptavidin plate, washed, reacted with an enzyme-conjugated antibody to digoxygenin, and developed with a chromogenic, fluorogenic, or chemiluminescent substrate for the enzyme. Alternatively, a radioactive method can be used to detect generated amplification products, for example, by including a radiolabeled deoxynucleoside triphosphate into the amplification reaction, then blotting the amplification products onto DEAE paper for detection. In addition, if one primer is biotinylated, then streptavidin-coated scintillation proximity assay plates can be used to measure the PCR products. Additional methods of detection can use a chemiluminescent label, for example, a lanthanide chelate such as used in the DELFIA^® assay (Pall Corp.), an electrochemiluminescent label such as ruthenium tris-bipyridy (ORI-GEN), or a fluorescent label, for example, using fluorescence correlation spectroscopy.

An integration junction also can be detected using an oligonucleotide probe that binds to the nucleotide sequence spanning the junction of the integrating polynucleotide, for example, a viral polynucleotide, and the target nucleic acid molecule. Such an oligonucleotide probe is designed to contain about six to ten nucleotides complementary to each of the integrating polynucleotide and target nucleic acid molecule sequences that span the integration junction (total length 12 to 20 nucleotides). Such an oligonucleotide probe, which is referred to as a "junctional probe" (see, for example, Tabak et al., Nucl. Acids Res. 9:4475-4483, 1981; Jonsson et al., Blood 76:2072-2079, 1990; Pongers-Willemse et al., Leukemia 12:2006-2014, 1998), lacks sufficient complementary for selective hybridization to only the integrating polynucleotide sequences or only the target nucleic acid molecule sequences, but can selectively hybridize to an integration product in which the sequences are covalently linked together.

The use of a junctional probe is particularly convenient where the end of the integrating polynucleotide comprising the integration junction is known and where the target nucleic acid molecule is a homopolymer (see Figure 8). Where the target nucleic acid molecule is comprises alternating nucleotides, for example, poly(dCdT), two junctional probes can be utilized such that hybridization occurs to a CATC junction and to a CACT junction, depending on whether the integrating polynucleotide integrates adjacent to a T residue or a C residue (see Figure 8B).

Where the target nucleic acid molecule comprises non-repetitive sequence such as a coding sequence of a gene or a randomly generated sequence, a junctional probe can comprise a sequence complementary to the appropriate sequence of the integrating polynucleotide and a sequence of about six to ten random nucleotides. Such a probe, which has characteristics of random primers used, for example, in cloning methods, can only hybridize when the random portion of the probe is complementary to the target nucleic acid sequence at the integration junction. Selective hybridization of a junctional probe can be detected using any of various methods, including, for example, a target amplification method such as PCR using a forward primer that is upstream in the viral cDNA (Saiki et al., Science 239:487-491, 1988), nucleic acid sequence based amplification (NASBA) using a similar forward primer (Compton, Nature 350:91-92, 1991), self-sustaining sequence replication (Fahy et al., PCR Meth. Appl. 1:25-33, 1991), transcription-mediated amplification (Kwoh et al, Proc. Natl. Acad. Sci.. USA 86:1173-1177, 1989), or strand displacement amplification (Vary, Nucl. Acids Res. 15:6883-97, 1987; Walker et al., Proc. Natl. Acad. Sci.. USA 89:392-396, 1992); a probe amplification method such as the ligase chain reaction (Wu and Wallace, Genomics 4:560-9, 1989; Barany and Gelfand, Gene 109:1-11, 1991), a Q-beta replicase mediated reaction (Lomeli et al, Clin. Chem. 35:1826-1831, 1989), cycling probe technology (CPT; Duck et al, BioTechniques 9:142-148, 1990), an Invader™ assay (Kwiatkowski et al, Mol. Diagnost. 4:353-364, 1999), or rolling circle amplification (Lizardi et al, Nat. Genet. 19:225-232, 1998); or a signal amplification reaction such as a branched chain DNA assay (Sanchez-Pescador et al., J. Clin. Microbiol. 26:1934-1938, 1988). The Invader™ assay, for example, can be used if the junctional-fragment probe has a mismatch at its 3' end, and is added along with a second oligonucleotide that binds just 5' to this region in the template strand to create a "flap", which allows detection using the Invader™ assay (Kwiatkowski et al., Mol. Diagnost. 4:353-364, 1999).

An oligonucleotide ligation reaction can be used to detect an integration junction. Such a reaction can use two oligonucleotides, including one that selectively hybridizes to a nucleotide sequence at the end of the integrating polynucleotide at the integration junction and the other that selectively hybridizes to a nucleotide sequence at the end of the target nucleic acid molecule at the junction. Upon hybridization of such oligonucleotides and contact of the reaction with a ligase, oligonucleotides that are adjacently hybridized are ligated, thereby identifying an integration junction.

A method of detecting integration of a polynucleotide into a target nucleic acid molecule can be performed, for example, by contacting a sample with a pre-integration complex (PIC), which includes at least an integrating polynucleotide and an integrase, and the target nucleic acid molecule, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule. After the integration events have been allowed to proceed for an appropriate amount of time, the effective concentration of target nucleic acid molecules in the sample is decreased, then the sample is contacted with a first amplification primer pair, under conditions that allow generation of a first amplification product from an integration product formed by the integrating polynucleotide of the PIC and the target nucleic acid molecule, and an amplification product is detected. The amplification product can be detected using any method as disclosed herein or otherwise known in the art, including for example, by further contacting the sample with a bilabeled oligonucleotide probe and detecting fluorescence due to degradation of the probe and unquenching of the fluorescent moiety; by performing a second amplification reaction, including, for example, a nested or hemi-nested PCR reaction, and detecting the second amplification product; or the like.

Decreasing the effective concentration of target nucleic acid molecules in the sample after the integration reaction has been allowed to proceed provides a means to optimize the assay. As used herein, the term "decreasing the effective concentration," when used in reference to a target nucleic acid molecule, means that the amount of intact target nucleic acid molecules in a unit volume of a sample is reduced relative to the amount prior the time the decreasing was performed. The effective concentration of target nucleic acid molecules can be decreased, for example, diluting the sample, or by contacting the sample with an exonuclease that degrades unreacted target nucleic acid molecules. As used herein, the term "unreacted target nucleic acid molecule" refers to a target nucleic acid molecule or portion thereof that does not contain an integration junction.

Where an integration assay utilizes an amplification reaction such as PCR, various modifications, including decreasing the effective amount of target nucleic acid molecules in the sample, can be used to optimize the sensitivity of the assay. Reduced sensitivity of an amplification reaction can occur, for example, due to the ability of a target nucleic acid molecule to sequester a polymerase such as Taq DNA polymerase or to sequester primers that hybridize thereto, resulting in each of the reactants being less available for the amplification of integration junctions and, where relevant, for degradation of a bilabeled oligonucleotide probe such as a TaqMan™ probe used in real-time PCR. Sequestration of a polymerase such as Taq can occur due to binding of the polymerase to the 5' ends of the target nucleic acid as a consequence of the Taq 5' exonuclease activity. As the number of 5' ends increases, so does the amount of polymerase bound thereto and, therefore, unavailable for an amplification reaction. Sequestration of the primers can occur due to hybridization of the primers to complementary sites in the target nucleic acid molecule that are not near integration sites. Such hybridization can further result in binding of the polymerase and, therefore, decreased availability of the polymerase for amplification. Another potential problem can arise when the primers bind to target sites that are not near an integration site, in which case the polymerase can extend the primer to the position of a second hybridized primer. At that point, a polymerase such as Taq, which has a 5' exonuclease activity, recognizes the boundary between the extension product and the second primer as a "nick," which acts as a substrate for the 5' exonuclease. As such, the exonuclease activity can degrade a primer that otherwise may have been extended to an integration junction.

Various general and specific solutions for minimizing or avoiding such problems, including methods for decreasing the effective concentration of target nucleic acid molecules in a sample, are provided herein. For example, binding of a polymerase such as Taq polymerase to the 5' ends of target nucleic acid molecules can be minimized by using a concatemeric form of the target nucleic acid molecule, which presents fewer 5' ends per mole of monomeric unit. The 5' ends also can be "capped", for example, by ligating an oligonucleotide incorporating a stem-loop structure, wherein the loop provides a sticky end that matches that of the target DNA but contains one or more 1,3-propanediol moieties ("C3 spacer") or other blocking residues such that it cannot serve as a "turn around" point for a reverse primer that is being extended using the target nucleic acid molecule as a template; or by otherwise making the 5' ends inaccessible, for example, by linking them to a solid substrate directly or indirectly such as through an amidite linkage.

The potential extension of primers that are bound to a target nucleic acid molecule at sites distant from an integration site can be minimized by reducing the length of an extension product that can be generated. One method for reducing such extension utilizes a triple helix-forming oligonucleotide such as a "PCR clamping" peptide nucleic acid (PNA), which can be designed so as to selectively hybridize to the 5' end of each monomeric unit at the elevated temperatures used for PCR. A target nucleic acid molecule also can be digested at the "head-to-tail" boundary of each monomer, then ligated to a linker containing one or more 1,3-propanediol moieties ("C3 spacer") at its center, thus blocking the extension of the reverse primer across the boundary of each monomeric unit. The strand of a target nucleic acid molecule that is recognized by the reverse primer also can be made discontinuous at the head-to-tail boundary of each monomer. By utilizing a discontinuity that is greater than about two nucleotides in length, and by blocking the 3' end, for example, by phosphorylation, the polymerase will not recognize the sequence as containing a nick and, therefore, will neither effect extension nor exonucleolytic cleavage. Potential 5' exonucleolytic digestion of a downstream primer that binds to a target nucleic acid molecule by the polymerase of an extending product can be minimized, for example, by incorporating one or a few 1,3-propanediol moieties ("C3 spacer") into the first few 5' nucleotides of the primer.

More general methods also are provided herein for optimizing an integration assay of the invention. For example, the reaction mixtures can be serially diluted following the integration reaction, then analyzed with single molecule sensitivity using a method such as hemi-nested PCR. Hemi-nested PCR can provide an ultrasensitive detection method, provided care is taken to minimize the introduction of errors due to the multiple dilutions and replicate amplification reactions that must be performed. Another general approach is to utilize a thermostable polymerase that lacks 5' exonuclease activity. Such DNA polymerases include, for example, from those isolated from Thermus "ubiquitous" (Hot Tub™), exonuclease-deficient DNA polymerases from Thermococcus litoralis (Vent™ exo-) and Pyrococcus sp. (Deep Vent™ exo-), Pyrococcus furiosis (Pfu, TurboPfu™), Thermatoga maritima (Ultma™), Thermoplasma acidophilum (ThermoSequenase™), Bacillus stearothermophilus large fragment DNA polymerase, and an exonuclease-deficient fragment from T. aquaticus (Stoffel Fragment), which can be obtained from commercial sources (Perkin-Elmer, Amersham, New England Biolabs). It should be recognized that the use of a polymerase lacking 5' exonuclease activity would preclude the use of a bilabeled oligonucleotide probe such as the TaqMan™ assay for detecting generation of the amplification product.

A useful method for optimizing the sensitivity of an integration method of the invention is to remove unreacted target nucleic acid molecules using an exonuclease such as λ exonuclease, which is a highly processive enzyme that degrades DNA starting at a 5' phosphate group, including, for example, starting at the phosphorylated, protruding 5' ends created by excision of the exemplified target nucleic acid molecule from a vector using Bam HI (Example 1). This method is particularly useful because binding of a PIC to a target nucleic acid molecule or the presence of an integration junction appears to protect DNA molecules containing the integration event from digestion by the λ exonuclease. Another advantage of using a 5' exonuclease is that such an exonuclease does not degrade amplification primers or detection oligonucleotides, which are not 5'-phosphorylated.

Sequence-specific degradation of unreacted target nucleic acid molecules also can be used to decrease the effective concentration of target nucleic acid molecules. Such degradation can be performed, for example, using a restriction endonuclease, particularly a Type I or Type III enzyme that can bind to the upstream portion in each monomer of a concatemerized target nucleic acid molecule, and cleave many bases downstream of the binding site. If the recognition sequence for the enzyme is in the 5' portion of each unit of the concatemer, then cleavage at the downstream locus will not occur if an integration event occurs because integration produces a gap in the DNA strand, thus preventing cleavage. As such, only unreacted target nucleic acid molecules, including unreacted monomers in a concatemerized target molecule are degraded. Since a target nucleic acid molecule can contain essentially any sequence, a desired restriction endonuclease recognition site readily can be introduced.

Ribozymes also can be used to degrade target nucleic acid molecules in a sequence-specific manner (Raillard and Joyce, supra, 1996). If a relatively short concatemer is utilized as a target nucleic acid molecule, the binding site for the ribozyme can be disrupted by the integration event, and only unreacted target nucleic acid molecules are degraded. Sequence-specific chemical nucleases also can be used to degrade unreacted target molecules. For example, nucleic acids or proteins that bind to a specific nucleotide sequence of the target molecule can be coupled to a 1,10-phenanthroline-copper complex, which degrades the sequence at a specific site (Francois et al., supra, 1989; Pendergrast et al, supra, 1994; Sigman et al., supra, 1993; Pan et al., supra, 1994; Perrin et al., supra, 1996). Again, if the concatemeric is sufficiently short, the binding site for the nucleic acid or protein coupled to the copper complex can be disrupted by the integration event, and only unreacted target nucleic acid molecules are degraded.

Dilution of the reaction mixtures prior to PCR detection also can reduce the amount of target nucleic acid molecules to a level that results in minimal spurious binding of polymerase or primers to the target molecule. However, dilution of the reaction mixtures also dilutes the integration junctions that are to be detected. To compensate for this, a linear (arithmetic) amplification (pre-amplification) can be performed using only a primer that binds to the integrating polynucleotide prior to the dilution step, then a method such as real-time PCR can be used to detect the integration events. Such a primer is designed to be complementary to the strand containing integration junction. A simple way for designing such a primer is to incorporate an additional sequence at or near the 3' end of a concatameric target DNA, then, following the integration reaction, performing a few cycles of a primer extension reaction using the 3' primer to generate multiple copies of the strand complementary to the integration junction. A PNA clamp directed against the additional sequence can be added to stop any further amplification from this 3' primer. A primer that hybridizes to the strand of the integrating polynucleotide complementary to the strand involved in the integration junction then can be added and about 10 to 100 cycles of linear, single primer amplification are performed. Where such a linear amplification step (pre-amplification) is performed, for example, for 10 cycles, and the reaction mixture is diluted 1:10, the excess target DNA is reduced 10-fold, whereas the linearly amplified strand containing the integration junction is not affected, resulting in a gain in sensitivity. Such a linear amplification step increases the amount of the nucleic acid molecules being amplified, and provides the additional advantage of maintaining their relative proportions (representation).

A similar result can be obtained by adjusting the melting temperature (Tm) of the primers and the temperature of the linear amplification steps. For example, if the 3' end primer is designed to have a Tm of 50°C, the initial cycles can use a thermocycling program with an annealing temperature of 45°C to create the strand complementary to the integration junction. Then, a forward primer with a Tm of 65 °C, for example, can be added for linear pre-amplification of the integration strand using a thermocycling program with an annealing temperature of 60°C, which is above the temperature that allows hybridization of the 3' end primer.

Another method for increasing sensitivity of an integration assay of the invention is to capture the integrating polynucleotides, including those comprising integration junctions, using, for example, a PNA duplex capture method (see, for example, Nielsen, Curr. Opin. Biotechnol. 12:16-20, 2001). This method utilizes two PNA "openers", which "open up" the duplexed DNA, and a capture PNA molecule, which binds to one of the strands in the opening, and provides a means to selectively collect all integrating polynucleotides in an integration reaction. Alternatively, since a gap exists at an integration site, the duplex can "breathe" and, therefore, is readily invaded by a triplex forming oligonucleotide or a hybridization capture oligonucleotide. Such a method provides a means to selectively enrich for nucleic acid molecules comprising integration junctions.

A method of the invention can be performed by contacting, for example, an aliquot of a sample containing PICs and integration buffer (without Mg⁺⁺), a test agent (if relevant), and the target nucleic acid molecules (and Mg⁺⁺), incubating at about 20°C to 37°C for 45 min to allow integration to occur; contacting the sample with λ exonuclease and incubating for about 37°C for 45 min; heat-inactivating the exonuclease enzyme at 75°C for 15 min; contacting the sample with a proteinase K solution, and incubating at about 60°C for 30 min; heat-inactivating the proteinase K at about 95°C for 15 min; transferring an aliquot of the reaction mixture to a solution containing, for example, Taq DNA polymerase, PCR primers, dNTPs, PCR buffer, and a detection agent such as a TaqMan™ real-time PCR probe, and performing an amplification reaction to detect an integration event. In such an aspect, the method requires several pipetting steps, and further requires transfer of an aliquot of the sample for the PCR reaction. By reducing the number of manipulations, however, the method can be adapted to a high-throughput screening format. In particular, a "homogenous" assay can be performed, wherein all of the reactions are performed in the same well or tube. For example, a sample to be examined can be added to a well and incubated under appropriate conditions until the signal can be read (a "mix-and- measure" or "mix-and-read" assay; see Sittampalam et al., Curr. Opin. Chem. Biol. 1 :384-391 , 1997). Such an assay format can be performed in less time and at a lower cost, is more reliable, and is particularly amenable to performing many assays, including screening many thousands of test agents.

To modify an integration assay of the invention for performing high throughput assays such as high throughput screening of test agents, for example, all of the reactants can be combined into an initial "master mix," which can be dispensed into the assay wells or other vessel in which the assays are to performed. The test agent is then simply added to the assay wells (i.e., a mix-and-measure format). The formulation of a master mix can be impeded due to the proteinase K and λ exonuclease, either of which can interfere with the components of the reaction mixture. For example, if proteinase K is included in a master mix, it can degrade PIC proteins such as integrase, as well as proteins needed for the assay such as Taq DNA polymerase. Similarly, if λ exonuclease is included in the master mix, it can degrade the target nucleic acid molecules prior to completion of the integration events. Potential problems due to proteinase K can be alleviated by including the proteinase K as a complex with an inhibitor such as the "double-headed" inhibitor of alpha-amylase and proteinase K that can be isolated from wheat germ (Roy and Gupta, Bioseparation 9:239-245, 2000) or as a complex with the inhibitory peptide sequence present in the C-terminus of lactoferrin (Singh et al., Proteins 33:30-38, 1998). After an incubation step to allow the PIC to integrate into the target DNA, the temperature can be raised to 60°C to dissociate the proteinase K from the inhibitor and activate its proteolytic activity, thus providing a "hot start" proteinase K reaction. For this format, the proteinase K subsequently is heat-inactivated at 95°C for 15 min, then Taq DNA polymerase is added to the same assay well (i.e., a homogenous, but not a mix-and-measure, format).

Alternatively, proteinase K can be omitted from the reaction, particularly if substantially purified PICs are contained in the initial master mix, such that the protein content in the original PIC extract is reduced. In an analogous system, treatment of MuA transposon reactions with a proteinase was not required in order to use PCR to analyze the joining of the transposon DNA with the target DNA to produce a "footprint" (Wei et al., EMBO J.16-7511-7520. 1997). PICs can be substantially purified using methods such as density-gradient ultracentrifugation, sedimentation velocity ultracentrifugation, or a combination thereof (Fassati and Goff, supra, 1999, 2001); isolation on a size-exclusion gel chromatography column (Farnet and Haseltine, supra, 1990; Miller et al. supra, 1997); affinity isolation by incorporating into the virions used to make the PICs a fusion protein of Npr (Wu et al., J. Virol. 69:3389-398, 1999) with an affinity tag such as the FLAG peptide sequence, which can be recognized by a specific antibody; the portion of protein A that binds to IgG; a sequence that can be biotinylated, which can bind to avidin or streptavidin; a polyhistidine sequence that can bind immobilized nickel ion; a calmodulin-binding peptide sequence; a chitin-binding domain; or combinations of such tags (Rigaut et al, Nat. Biotechnol. 17:1030-1032, 1999); or by treating PIC- containing extracts with a fusion protein consisting of a tag (as above) covalently linked to a PIC-interacting motif such as the sequence in uracil DNA glycosylase containing the WXXF (SEQ ID NO: 18) motif (BouHa dan et al., J. Biol. Chem. 273:8009-8016, 1998; Turelli et al., Mol. Cell 7:1245-1254, 2001, each of which is incorporated herein by reference), followed by affinity chromatography to isolate the PICs. An advantage of removing proteinase K from the assay entirely is that all of the protein components of a reaction, including, for example, PIC proteins, λ exonuclease, and Taq DNA polymerase, can be contained in a single initial master mix, thus facilitating the preparation of a mix-and-measure assay format.

λ exonuclease also can be included a master mix if its exonuclease activity is controlled. Since λ exonuclease is a 5'-to-3' exonuclease that only degrades DNA strands containing a 5' phosphorylated end, primers and their extension products are not susceptible to degradation. However, the target DNA is specifically designed to contain 5' phosphorylated ends such that it can be degraded by λ exonuclease. To control the activity of λ exonuclease, its concentration can be reduced so that a longer time (e.g., 2 hr) is needed to substantially reduce the concentration of target DNA in the reaction at an appropriate incubation temperature. PICs integrate into target DNA very rapidly, typically with about 90% efficiency within 15 min of contact with the target DNA (Brown et al, Cell 49:347-356, 1987). Consequently, if a reduced concentration of λ exonuclease is incorporated into the initial master mix along with the PICs, PCR primers, and target DNA, and maintained at about 4°C until time to perform an assay, for example, until a test agent is added to the assay, then the λ exonuclease degradation of the target DNA will largely follow the integration of the PICs into the target DNA. Such a format, combined with the elimination of proteinase K discussed above, can be used to establish a mix-and-measure assay for PIC integration.

The present invention also provides methods of identifying an agent that modulates integration of a polynucleotide into a target nucleic acid molecule. Such a method can be performed, for example, by contacting an integrating polynucleotide, a target nucleic acid molecule, and a test agent, under conditions that, in the absence of the test agent, allow integration of the polynucleotide into the target nucleic acid molecule; and detecting a change in the number of integration events of the polynucleotide and the target nucleic acid molecule in the presence of the test agent as compared to the number of integration events in the absence of the test agent, wherein a change identifies the test agent as an agent that modulates integration of the polynucleotide into the target nucleic acid molecule.

As used herein, the term "modulates," when used in reference to the effect of an agent on integration of a polynucleotide into a target nucleic acid molecule, means that the agent that can reduce or inhibit the number of integration events effected by an integrating polynucleotide, or can increase the number of integration events. Such a reduction or inhibition or an increase can be detected using the methods disclosed herein, including, for example, by performing assays in parallel, wherein the samples lack or contain a test agent, or a control agent known to modulate integration, for example, an integrase inhibitor such as purpurin, quinalizarin or a di-keto compound such as L-731,988 or L-708,906 (Hazuda, supra, 2000). Generally, though not necessarily, a series of samples containing varying amounts of a test agent are examined such that an effective amount for modulating integration can be identified and a dose response effect, if such a response occurs, can be detected.

An agent can act with respect to any component involved in the integration event, including, for example, on the integrating polynucleotide, the integrase or other enzyme involved in a cleavage or ligation reaction associated with the integration event, or a viral or cellular factor involved in the integration event. An agent that can reduce or inhibit integration activity can be useful, for example, for minimizing adverse genetic effects due to the action of a mobile genetic element, including, for example, for reducing or inhibiting retrovirus integration into a mammalian genome, or transposon integration into plant cell genome, or the like. An agent that can increase integration activity can be useful, for example, for increasing the likelihood of integration of a retroviral vector into a cell that may be refractory to such integration such as a non-dividing cell, or of a transposon such that mutant plant cells, for example, can be obtained and examined for a desirable characteristic, or as a research tool for generally increasing the mutation rate of an organism such as

Drosophila or cells of an organism, since the integrating polynucleotide can further act as a tag for identifying the genomic sequence associated with the mutation. A test agent can be any type of molecule, including, for example, a peptide; a polynucleotide; a derivative of a peptide or polynucleotide such as a peptide nucleic acid, which is a nucleic acid molecule containing one or more peptide bonds linking the nucleotide monomers; a small organic molecule such as a peptidomimetic; and the like. In one embodiment, the test agent comprises one of a library of test agents, in which case the method can, but need not, be performed in a high throughput format. The library of test agents can be any type of library, for example, a library of randomly generated, biased, or variegated molecules (see, for example, U.S. Pat. Nos. 5,264,563; 5,837,500; and 5,962,219). Methods for preparing a combinatorial library of molecules that can be tested for the ability to modulate integration are well lαiown in the art and include, for example, methods of making a phage display library of peptides, which can be constrained peptides (see, for example, U.S. Pat. Nos. 5,622,699 and 5,206,347; Scott and Smith, Science 249:386-390, 1992; Markland et al, Gene 109:13-19, 1991; each of which is incorporated herein by reference); apeptide library (U.S. Pat. No. 5,264,563); a peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14:83-92, 1995; a nucleic acid library (O Connell et al, Proc. Natl. Acad. Sci.. USA 93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Gold et al, Ami. Rev. Biochem. 64:763-797, 1995); an oligosaccharide library (York et al., Carb. Res.. 285:99-128, 1996; Liang et al., Science. 274:1520-1522, 1996; Ding et al, Adv. Expt. Med. Biol.. 376:261-269, 1995); a lipoprotein library (de Kruif et al, FEBS Lett.. 399:232-236, 1996); a glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol., 130:567-577, 1995); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordon et al, J. Med. Chem.. 37:1385-1401, 1994; Ecker and Crooke, Bio/Technology, 13:351-360, 1995). Polynucleotides can be particularly useful as agents that can modulate integration of a polynucleotide into a target nucleic acid molecule because nucleic acid molecules having binding specificity for cellular targets, including cellular polypeptides, exist naturally, and because synthetic molecules having such specificity can be readily prepared and identified (see, for example, U.S. Pat. No. 5,750,342). Accordingly, the present invention also provides an agent identified by such a method, such agents being useful, for example, in the preparation of a medicament for treating a disorder associated with an integrating polynucleotide, for example, infection by a retrovirus such as HIV.

In another embodiment, a method of the invention can be used to identify an agent that modulates the activity of a pro-preintegration complex ("pro-PIC").

Integration of a retroviruses generally requires that the host cell is activated (Varmus et al., Cell 11. 2:307-319, 1977). While lentiviruses such as HIV-1 (Lewis and Emerman, J. Virol. 68. 1 :510-516, 1994) and lentiviral vectors derived from HIV-1 (Naldini et al., Science 272:263-267, 1996) can integrate in resting host cells under certain circumstances, HIV-1 generally cannot integrate into the genome of human T lymphocytes (Stevenson et al, EMBO J. 9:1551-1560, 1990; Korin and Zack, J. Virol. 73:6526-6532, 1999; Wu and Marsh, Science 293:1503-1506, 2001). Reverse transcription of HIV-1 RNA into cDNA can occur in resting T lymphocytes, but the viral DNA remains in the cytoplasm in a "preintegration latency" state (Pierson et al., Ann. Rev. Immunol. 18:665-708, 2000).

Cytoplasmic (unintegrated) HIV-1 DNA has been found in T lymphocytes taken from patients with asymptomatic or early HIV-1 infection (Bukrinsky et al, Science 254:423-427, 1991; Chun et al., Nature 387:183-188, 1997; Blankson et al., J. Infect. Dis. 182: 1636- 1642, 2000), and in macrophages present in the brains of subjects with HIV-1 encephalitis (Pang et al., Nature 343:85-89, 1990). Similarly, lentiviral vectors based on HIV-1 generally cannot effectively transduce resting human CD4⁺ T cells unless the cells are first treated with a cocktail of activating cytokines such .as interleukin-2 (IL-2), IL-7 and IL-15 (Unutmaz et al, J. Exp. Med. 189:1735-1746, 1999). In two studies, activation of the MEK/ERK signal transduction pathway in resting human CD4+ T cells was required for HIV-1 to integrate (Popik and Pitha, J. Virol. 74:2558-2566, 2000; Marozsan et al., J. Virol. 75:8624-8638, 2001).

As disclosed herein (Example 2), a method of the invention was used to determine that no integration-competent PICs were detectable in cells infected with an HIV-1-based lentiviral vector (Chinnasamy et al., Blood 96:1309-1316, 2000, which is incorporated herein by reference; Naldini et al., supra, 1996), which can undergo reverse transcription in resting human blood lymphocytes in culture. In contrast, when the human blood lymphocytes were preactivated using phytohemagglutinin (PHA) and IL-2 (Coligan et al, "Current Protocols in Immunology" (ed. R. Coico; John Wiley & Sons, Inc., New York 2001),which is incorporated herein by reference), fully integration-competent PICs were detected. Similarly, if the human blood lymphocytes were first infected, while in a resting state, with the lentiviral vector construct, then activated by stimulation with anti-CD3 and anti-CD28 antibodies, integration-competent PICs were detected. These results demonstrate that a precursor the preintegration complex, i.e., a pro-PIC, is formed following reverse transcription, but is not integration competent.

A method of the invention provides a means to identify pro-PICs. For example, a sample can be examined for the presence of a retrovirus by detecting retroviral cDNA, for example, using a real-time PCR (TaqMan™) analysis (Rossio et al., J. Virol. 72:7992-8001, 1998, which is incorporated herein by reference). Upon detecting the presence of retrovirus, the sample then can be examined for integration- competent PICs using a method of the invention, wherein the ratio of integration- competent PICs to a retroviral intermediate, for example, reverse transcribed cDNA, provides a measure of the formation of pro-PICs. As such, the method also provides a means for determining the stage in the replication cycle of a retrovirus, wherein the retrovirus is unable to integrate into a host cell genome. If desired, a method of identifying a pro-PIC can further include isolating the pro-PIC, for example, by subjecting an extract from a retrovirus infected cell to a physical separation method such as density gradient ultracentrifugation and/or sedimentation velocity ultracentrifugation (Fassati and Goff, J. Virol. 73:8919-8925, 1999; Fassati and Goff, J. Virol. 75:3626-3635, 2001). Accordingly, the present invention provides isolated pro-PICs.

The present invention also provides a method of identifying an agent that modulates the activity of a pro-PIC. For example, a method of the invention can be performed using pro-PICs as the integrating polynucleotide and, upon contact with a test agent, integration activity can be measured. The detection of integration events in the presence of a particular test agent (as compared to in the absence of the agent or above a base line level that occurs in the absence of the agent) thus identifies an agent that renders a pro-PIC integration competent. Such an agent can be useful, for example, in combination with a gene therapy method using a retroviral vector, since the agent can facilitate integration of the retroviral vector in a cell such as a non- dividing cell, which otherwise may be refractory to integration. A method of the invention also can be used to identify an agent that reduces or inhibits the ability of a pro-PIC to become integration-competent. Such an agent being useful, for example, to treat or prevent retroviral infection, for example, an HIV-1 infection, where a substantial proportion of the reservoir of latent HIV-1 can be present in a pre- integration latency stage in infected individuals (Pierson et al., supra, 2000). To the extent that pro-PICs represent the physical embodiment of pre-integration latency, an agent that disrupts the ability of pro-PICs to become integration-competent can be useful for therapeutic purposes, including in the preparation of medicaments for modulating integration of a retroviral genome into a host cell genome.

The present invention also provides methods of identifying a factor that mediates integration of a polynucleotide into a target nucleic acid molecule. As used herein, the term "factor that mediates" or "factor involved in," when used in reference to integration of a polynucleotide into a target nucleic acid molecule, means any molecule that is required for or facilitates such integration. Thus, the factor can be a synthetic or naturally occurring polypeptide or nucleic acid molecule, including, for example, a protein, nucleic acid or other viral or cellular macromolecule, or a portion thereof having the requisite activity.

A method of identifying a factor involved in integration of a polynucleotide into a target nucleic acid molecule can be performed, for example, by contacting the polynucleotide and the target nucleic acid molecule with a test factor, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule; and detecting integration of the polynucleotide into the target nucleic acid molecule. The integrating polynucleotide included in the sample can be a naked polynucleotide; can include an integrase, transposase, recombinase, or other relevant enzyme involved in integration associated therewith; or can be associated with one or more other cellular factors or viral factors, which can, but need not include the relevant enzyme. For example, the polynucleotide can comprise a retroviral LTR; or a retroviral LTR and retroviral integrase associated therewith; or a PIC; or a salt- stripped PIC. The test factor can be a synthetic molecule such as a peptide, polynucleotide, or the like, which can be one of a library of molecules; or can be naturally occurring, for example, a cellular factor such as a cellular protein, proteolipid, glycoprotein, polynucleotide, nucleoprotein, or the like, which can be in an isolated form or in the form of a cellular or viral extract or a fraction of such an extract obtained by a chromatography or electrophoresis method or the like. Synthetic molecules to be used as a test factor, including libraries of such molecules, can be prepared using the methods as described for producing libraries of test agents (see above).

A method of the invention was exemplified by identifying cellular factors involved in integration of an HIV viral genome into a target nucleic acid molecule (Example 1). The cellular factors were identified by contacting salt-stripped PICs obtained from HIV infected cells and a target nucleic acid molecule with an extract obtained from uninfected T cells that are susceptible to HIV infection. Integration activity of the salt-stripped PICs was obtained upon addition of the cellular extract (Figure 6), thus demonstrating that a method of the invention can be used to identify factors involved in integration.

A method of identifying a factor that mediates integration of a polynucleotide into a target nucleic acid molecule can further include a step of isolating the factor. As used herein, the term "isolating" or "isolate," when used in reference to a cellular factor or viral factor, means that the factor is in a form other than that in which it occurs in nature. Generally, an isolated factor such as a polypeptide factor can be identified as a discrete band, which can be the only band or one of several bands, following electrophoresis through a gel such as a polyacrylamide gel. A factor that mediates integration can be isolated using any method routinely used for isolating the particular type of molecule comprising the factor, i.e., whether the factor is a polypeptide, polynucleotide, small organic molecule, or the like. For example, the cellular factors contained in the T cell extract that are involved in integration activity can be isolated using a method such as ammonium sulfate fractionation, high performance liquid cliromatography or other chromatographic method, or capillary electrophoresis or other electrophoresis method, and the fractions obtained can be examined for the presence of factors that mediate integration. Accordingly, the present invention provides an isolated factor obtained using such a method.

The present invention further provides a method of generating a linear amplification product of a selected strand of a nucleic acid molecule, including a method of using a self-suppressing oligonucleotide primer to generate the linear amplification product. A linear amplification method can be performed, for example, by contacting a sample containing the nucleic acid molecule with at least one oligonucleotide primer that selectively hybridizes to the selected strand, under conditions that allow generation of a linear amplification product comprising the oligonucleotide primer, wherein the oligonucleotide primer can form a secondary structure, which prevents the primer from selectively hybridizing to the selected strand, and wherein the conditions that allow generation of the linear amplification product include conditions above the melting temperature of the secondary structure of the oligonucleotide primer. Such a primer, which is designed such that it can form a secondary structure such as a stem-loop structure below a predetermined melting temperature, is referred to herein as a "self-suppressing primer" or "self-suppressing oligonucleotide primer." Considerations for designing a self-suppressing primer are similar to those for determining conditions that allow selective hybridization, for example, of a primer to a target nucleic acid molecule (see above).

The self-suppressing primer can selectively hybridize at any position on the selected strand, provided that extension of the primer and linear amplification can occur, including at or near a 3' terminus of the nucleic acid molecule. The nucleic acid molecule to which the primer hybridizes can be single stranded, for example, deoxyribonucleic acid strand complementary to an mRNA (i.e., a cDNA), in which case the single strand is the selected strand, or can be a double stranded nucleic acid molecule, which includes a first strand and a second strand, either of which can be the selected strand for linear amplification. In one embodiment, the nucleic acid molecule is a double stranded DNA that contains a nick in one strand, the second strand is the selected strand, and the linear amplification product generated therefrom spans the nick. In another embodiment, the selected strand includes an integration junction, which is formed by integration of a polynucleotide into a target nucleic acid molecule.

The method of generating a linear amplification product can further include using the linear amplification product as a template to generate an exponential amplification product. Such a method can be performed, for example, by contacting the linear amplification product with an amplification primer pair, which includes a first primer that selectively hybridizes to a nucleotide sequence of the selected strand of the nucleic acid molecule, and a second primer that selectively hybridizes to a nucleotide sequence of the linear amplification product, under conditions that allow generation of an amplification product from the amplification primer pair, wherein such conditions include conditions that are below the melting temperature of a secondary structure formed by the oligonucleotide primer, thereby preventing the oligonucleotide primer from selectively hybridizing to the selected strand.

The method of generating a linear amplification product can further include detecting generation of the linear amplification product. Such detecting can be performed using any method as disclosed herein or otherwise known in the art, for example, by contacting the sample with a bilabeled oligonucleotide probe, which comprises a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the selected strand that is in a 3' position with respect to the oligonucleotide primer; and detecting fluorescence due to the fluorescent moiety. Such a detecting method similarly can be used to detect generation of an amplification product, in a method in which the linear amplification product is used as a template for an amplification reaction such as PCR, for example, by contacting the sample containing the amplification primer pair with a bilabeled oligonucleotide probe, which can selectively hybridize to a nucleotide sequence of the amplification product; and detecting fluorescence due to the fluorescent moiety.

The nucleic acid molecule containing the selected strand can be an isolated nucleic acid molecule that is added to a reaction mixture for performing a method of the invention. The sample, which contains the nucleic acid molecule, also can be all or a portion of a naturally occurring sample, for example, a cell sample, which is obtained from a subject using a method such as a biopsy procedure. The subject can be any subject for which it is desired to generate a linear amplification product of a selected strand of a nucleic acid molecule, which can be a nucleic acid molecule normally present in the cell such as a portion of genomic DNA or plasmid DNA; or that can be derived from a naturally occurring nucleic acid molecule, for example, a cDNA derived from an mRNA; or that comprises an exogenous nucleic acid molecule that has been introduced into the cells, for example, by infection. Generally, the subject is a vertebrate subject such as a mammalian subject, for example, a human subject.

The linear amplification method as disclosed herein provides a convenient and efficient means for analyzing gene expression because the method results in proportional amplification, thus providing a product that is quantitatively representative of the starting material. Accordingly, the method can be performed starting with a very small sample such as that obtained, for example, by a needle biopsy. As such, a method of linear amplification can be useful for analyzing and comparing populations of mRNA molecules expressed in cells or tissues, including for comparing such expression in normal cells and cells obtained from a subject suffering from a pathologic condition, or for comparing expression in cells at different stages of development or differentiation, and the like.

One method for detecting and comparing such mRNA populations (referred to as a "transcriptome") is through the use of DNA microarrays (Brown and Botstein, Nat. Genet. 21:1 Suppl:33-37, 1999). For example, the response of tumor cells to a particular chemotherapy can be deduced by transcriptome analysis (Alizadeh et al., Nature 403:503-511, 2000; Scherf et al., Nat. Genet.24:236-244, 2000). Tumors, such as breast cancers also can be subtyped by gene expression analysis (Perou et al., Nature 406:747-752, 2000). Since many cancers are diagnosed using minimally invasive techniques such as fine needle biopsy, which do not yield much tissue for analysis, more sensitive methods of gene expression analysis are needed (Emmert-Buck et al, Am. J. Pathol. 156:1109-1115, 2000). Various methods have been reported for such analysis (Brady, Yeast 17. 3:211-217, 2000). For example, Eberwine et al. described a method for reverse transcribing small amounts of RNA with a primer containing an RNA polymerase promoter, which allows the production of about 1,000 copies of cRNA from each cDNA molecule (Eberwine et al, Proc. Natl. Acad. Sci., USA 89:3010- 3014, 1992). Belyavsky et al. described a way of adding a 5' primer binding site to a cDNA by using terminal transferase, followed by PCR to amplify the cDNA

(Belyavsky et al., Nucl. Acids Res. 17:2919-2932, 1989). Brady and Iscove used a somewhat different 5' terminal transferase tailing method to create a 5' primer-binding site for PCR amplification , a process called PolyAPCR (Brady and Iscove,. Meth. Enzymol. 225:611-263, 1993). Dixon et al. used both unique and degenerate sequences to amplify cDNA in a technique called TPEA (Dixon et al, Nucl. Acids Res. 26. 19:4426-4431, 1998). However, none of these methods have become completely accepted as reliable means of working with small amounts of RNA without losing the representation of the sequences in the original sample.

The present invention provides a method of linear amplification for generating cDNA useful for a gene expression analysis. Such a method can be performed, for example, by contacting polyadenylated mRNA, for example, containing 17 adenosines, with an "anchored" oligo-dT polynucleotide, which contains on its 5' end a "heel" sequence, for example, the anchored polynucleotide 5'-CTCTCAAGGATCTTACCGCTTTTTTTTTTTTTTTTT(A,G,C -3' (SEQ ID

NO:19; Dixon et al., supra, 1998). The choice of a primer mixture that contains an A, G, or C on its 3' end serves to "anchor" this primer to the extreme 5' end of the poly(A) tract. The "heel" sequence (underlined) creates a binding site for the linear amplification primer in every cDNA molecule produced. The cDNA strand generated using the anchored oligo-dT polynucleotide is converted to a double-stranded DNA molecule, and linear amplification is used to increase the number of cDNA molecules present. Briefly, a primer oligonucleotide complementary to the heel is added with a thermostable DNA polymerase such as Taq polymerase and dNTPs. The mixture is then thermocycled so that in each cycle one new strand is synthesized from each template molecule. Typically, 30 to 100 cycles are used. The linearly amplified cDNA then can be analyzed by any of detection method as disclosed herein or otherwise lαiown in the art. Because the product of linear preamplification is single stranded DNA, it can be examined directly, for example, using a DNA microarray containing probe oligonucleotides or cDNA molecules of interest.

The present invention also relates to a kit, which contains one or more reagents useful for practicing a method of the invention. As such, a kit of the invention can contain, for example, a target nucleic acid molecule; at least one primer, which can selectively hybridize to a nucleotide sequence of the target nucleic acid molecule and, if desired, can be a primer of an amplification primer pair; and an exonuclease. The target nucleic acid in the kit can be about 10 to 1000 nucleotides in length, generally about 50 to 500 nucleotides in length, and particularly about 100 to 250 nucleotides in length, and can be provided as a monomer, or as a concatemer of about 2 to 1000 monomeric target nucleic acid molecules, generally about 10 to 250 monomeric target nucleic acid molecules, and particularly about 25 to 100 monomeric target nucleic acid molecules; or can include a combination of monomeric and concatemerized target nucleic acid molecules, including monomers of varying length or concatemers containing various numbers of linked monomeric units.

A kit of the invention also can contain a polynucleotide that can integrate into the target nucleic acid molecule supplied with the kit. In various embodiments, the polynucleotide that can integrate can comprise an isolated PIC, an isolated pro-PIC, a salt-stripped PIC, or a combination thereof. Such an integrating polynucleotide can be useful, for example, as a control to confirm or normalize results of an integration assay performed using the kit. Such a kit also can contain a second primer of an amplification primer pair, wherein the second primer selectively hybridizes to a nucleotide sequence of the integrating polynucleotide. In addition, the kit can contain an integrase that can mediate integration of the polynucleotide into a target nucleic acid molecule. A kit of the invention also can contain, for example, a target nucleic acid molecule; at least one primer, which can selectively hybridize to a nucleotide sequence of the target nucleic acid molecule and, if desired, can be a primer of an amplification primer pair; and a polynucleotide that can integrate into the target nucleic acid molecule. Such a kit also can contain a second primer of an amplification primer pair, wherein the second primer selectively hybridizes to a nucleotide sequence of the integrating polynucleotide.

In addition, a kit of the invention can contain a detector oligonucleotide, which can selectively hybridize to a nucleotide sequence of a recombinant nucleic acid molecule formed upon integration of an integrating polynucleotide into the target nucleic acid molecule; or can selectively hybridize to an amplification product generated therefrom. The detector oligonucleotide can be labeled with a detectable moiety, and can be a bilabeled oligonucleotide containing a fluorescent moiety and a quenching moiety, which, when in proximity to the fluorescent moiety, quenches fluorescence. Alternatively, the kit can contain an unlabeled detector oligonucleotide, and can further include one or more moieties and reagents for labeling the oligonucleotide, including, for example, a variety of fluorescent moieties such that a fluorescent moiety useful for a particular application can be selected, as desired. The kit also can contain at least one primer of a second amplification primer pair, wherein the primer can selectively hybridize to a nucleotide sequence of an amplification product generated using the first amplification primer pair.

A kit of the invention also can contain an oligonucleotide primer, which can selectively hybridize to a selected strand of a nucleic acid molecule under predetermined conditions, but which forms a secondary structure that reduces or inhibits the ability to selectively hybridize to the selected strand under other conditions. Such a kit also can contain an amplification primer pair, wherein the primers of the amplification primer pair can selectively hybridize to a target nucleic acid molecule and provide substrates for amplification under condition in which the oligonucleotide primer forms the secondary structure. A kit of the invention can further include a master mix containing all of the component for performing an integration assay, including an integrating polynucleotide, for example, PICs, pro-PICs or salt-stripped PICs, a target nucleic acid molecule, and appropriate buffers for performing the integration reaction. Such a master mix also can contain λ exonuclease, and can further include reagents for detecting an integration event, for example, a linear amplification primer, an amplification primer pair, a detector oligonucleotide such as a bilabeled oligonucleotide probe, or the like. Such a kit can be useful for performing a high throughput assay in an array format on a solid support such as a chip, glass slide, bead, or the like, for example, a high throughput assay for screening test agent to identify an agent that modulates integration of a polynucleotide into a target nucleic acid molecule, or a high throughput assay for identifying factors involved in integration.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE 1 NUCLEIC ACID INTEGRATION ASSAY This example provides a method for detecting integration of a viral nucleotide sequence into a target nucleic acid molecule.

A. METHODS

Integration substrates Plasmid DNA containing head-to-tail concatemers encoding for drag-line silk protein was as described (Prince et al, Biochemistry 34:10879-10885, 1995; Winkler et al. Biochemistry 39:12739-12746, 2000, each of which is incorporated herein by reference). Plasmid DNA was isolated from transformed E. coli DH5α and purified using a Maxi Prep Kit (Qiagen Inc.; Valencia CA). The insert in the plasmid contained monomeric units of 105 base pairs (bp) repeated 32 times. Insert DNA was prepared by overnight digestion with Bam HI, which creates the protruding 5' ends that are a substrate for λ exonuclease, purified by electrophoresis in agarose, and isolated using a QIAquick Gel Extraction Kit (Qiagen). To prepare single monomeric units of the 105 bp sequence, insert DNA was digested with Sty I, which cleaves between each unit, and gel purified.

Oligonucleotide primers

The real-time PCR (TaqMan™) detection of integration junctions used the following oligonucleotides: forward primer LTRTaq5: 5'-GTGTGTGCCCGTCTGTTGTG-3' (SEQ ID NO:l); reverse primer SILKREVla: 5'-CAGCACCGCCCATTGC-3' (SEQ ID

NO:2); and probe LTRTaqP, 5'-FAM-CTGGTAACTAGAGATCCCTCAGACCCTTT- TAGTCAG -TAMRA-3' (SEQ ID NO:3; TriLink Biotechnologies, Inc.; San Diego CA; "FAM" - 6-carboxyfluorescein; "TAMRA" - tetramethylrhodamine).

In some experiments, integration reactions were further amplified by hemi-nested PCR using the following oligonucleotides:

HIVout: 5'-CAATAAAGCTTGCCTTGAGTGC-3' (SEQ ID NO:4); and HIVin2; 5'-AGTAGTGTGTGCCCGTCTGTTGTG-3' (SEQ ID NO:5).

Preparation of PIC-containing cytoplasmic extracts

PICs were prepared from the detergent lysates of cells acutely infected by HIV-IL_AI (LAV-1 strain; Farnet and Haseltine, Proc. Natl. Acad. Sci. USA 87:4164- 4168, 1990; Ellison et al, J. Virol. 64:2711-2715, 1990, each of which is incorporated herein by reference). Briefly, CEM cells were infected by HIV-I_LA_I under BSL-3 conditions and cultured for 7-10 days until cytopathic changes (ballooning and apoptosis) were evident in the majority of cells by phase microscopic examination (Terai et al., J. Clin. Invest. 87:1710-1715, 1991, which is incorporated herein by reference). The cells were then counted, and co-cultured with a 10-fold excess of SupTl cells (American Type Culture Collection; Manassas VA) in RPMI 1640 with 10% fetal bovine serum at 5% CO in a flask placed on end so that the infected CEM cells and the uninfected SupTl cells were in contact (U.S. Pat. Nos. 5,759,768 and 6,218,181, each of which is incorporated herein by reference).

After 5 to 7 hr, when the cultures consisted of about 80% syncytia just large enough to be visible to the unaided eye, the cells were pelleted by centrifugation at 160 x g for 5 min. The cells were washed once by gently resuspending them in 10 mM Tris-HCl (pH 7.4), 150 mM KC1, 5 mM MgCl₂ and 20 μg/ml aprotinin (Buffer A; Ellison et al., supra, 1991), then pelleted by centrifugation. Based on the original cell counts of the culture, the pellet was resuspended in Buffer A plus 0.025% digitonin (Calbiochem-Novabiochem Corp.; San Diego CA) at a concentration of 2 x 10⁷ cells/ml for 10 min at room temperature. The nuclei in the cell lysate were pelleted at 1,000 x g at 4 °C for 3 min in an Avanti 30 centrifuge with a F2402H rotor (Beckman Coulter Inc.; Fullerton CA).

The post-nuclear supernatant was transferred to a new microtube and the remaining cellular debris was removed by centrifugation at 8,000 g at 4°C for 10 min. Forty percent sucrose was added to the supernatant to a concentration of 8% sucrose final, and the PIC-containing cytoplasmic extract was aliquoted, snap frozen in liquid nitrogen, and stored at -80°C. PIC activity was stable at this temperature for at least six months. Typically, the extracts contained approximately 0.8 mg/ml of total protein measured using the BCA reagent (Pierce Chemical Corp.; Rockford IL), and 0.5-2.0 x 10⁸/ml copies of HIV-1 cDNA as quantified by real-time fluorescence- monitored PCR using LTR-specific primers and TaqMan probe (Rossio et al., supra, 1998).

In vitro integration reactions

Integration reactions (15 μl total volume) were prepared on ice in 200 μl thin- walled PCR tubes (either in 8 tube strips or in 96-well plates) by adding 5 μl of a master mix consisting of 1 μl of target DNA (3.3-100 ng/ μl, with 11-33 ng/ μl of S32 concatemer being optimal for general use), 1.5 μl of 10X integration buffer (200 mM HEPES, pH 7.4, 50 mM MgCl₂, 10 mM DTT), and 2.5 μl of 30 % PEG-8000 (Sigma Chemical Corp.; St. Louis MO). Ten μl of PIC-containing cytoplasmic extract was added and the tube contents were mixed by pipetting up and down. The tubes were kept on ice and placed in a thermocycler (GeneAmp 9600, Applied Biosystems; Foster City CA) and incubated at 4 °C for 10 min, 37°C for 45 min, and then at 60°C for 5 min.

Post-reaction processing

Unreacted target DNA was removed by adding 3-5 units of λ exonuclease in 15 μl of a 2X concentration of its supplied buffer (New England Biolabs; Beverly MA). The tubes (now containing 30 μl) were returned to the thermocycler and incubated at 37°C for 45 min, which allows the λ exonuclease to degrade the target DNA, followed by 75°C for 10 min to heat-inactivate the λ exonuclease. The processing was completed by adding 20 μl of proteinase K (1 mg/ml in water) and incubating in the thermocycler at 60°C for 30 min, followed by heat inactivation at 95°C for 15 min. The processed integration reactions (50 μl total volume) could be stored at 4°C for at least one week prior to TaqMan™ analysis. In some experiments, processed and unprocessed reactions were analyzed by polyacrylamide gel electrophoresis, staining with SYBR Gold (Molecular Probes, Eugene, OR), and visualization with UV light.

Real-time fluorescence-monitored PCR detection of integration junctions

In some experiments, the λ exonuclease- and proteinase K-processed integration reaction mixtures were diluted 10-fold in water, then analyzed by real-time PCR. For integration reactions containing colored inhibitors, the inhibitor was removed by concentration/filtration of the processed integration reaction mixture using Microcon PCR 96-well plates according to manufacturer's protocol (Millipore; Bedford MA).

TaqMan™ reactions were set up in optical grade 96 well thermocycler plates by adding 20 μl of a master mix containing 12.5 μl of TaqMan™ Universal PCR Master Mix 2X (Applied Biosystems), 2.5 μl each of 9 μM LTRTaq5 and

SILKREVla amplification primers (900 nM final concentration) and 2.5 μl of 2 μM LTRTaq5P probe (200 nM final concentration). Five μl of the processed integration reaction was added to each well, and the tube contents were mixed by pipetting up and down. Real-time fluorescence-monitored PCR reactions were performed on an Applied Biosystems Model 7700 Sequence Detection System. The temperature profile for the reaction was 50°C for 2 min; 95°C for 10 min; and then 95°C for 15 sec and 60°C for 1 min for 45 cycles.

Using the manufacturer's software, the cycle number at which fluorescence exceeded background (C_t) was determined for each well. For each real-time PCR analysis, a standard curve was generated using dilutions in water of a cloned integration junction (below) calculated to provide 3, 10, 100, and 1000 integration junctions per well. The reactions were set up as 2-fold or 3 -fold replicates and typically differed by less than 0.4 C_t.

Data analysis of the integration assay

To deduce the number of integration junctions in each sample, the C_t value for the sample was compared to the cloned integration junction standard curve following linear regression analysis. Because each TaqMan™ reaction used only 5 μl of the 50 μl processed integration reaction, the number of integration junctions was multiplied by 10 to arrive at the number of integration events produced by the original 10 μl of PIC-containing cytoplasmic extract. If the processed reaction was diluted prior to TaqMan™ analysis, an additional correction was made for the dilution factor. No correction was made, however, for the presumably equal number of integrations into the complementary, unmeasured strand of the target DNA. Statistical calculations were performed using the InStat software program (GraphPad Software Inc., San Diego, CA).

Hemi-nested PCR

Hemi-nested PCR was used to isolate PCR products in order to confirm that the method identifies integration junctions. For the first round of PCR, integration reactions were diluted 1:1,000 in water and 5 μl was amplified in a 25 μl reaction using the HINout and SILKREVla primers, and HotStarTaq (Qiagen) according to the manufacturer's instructions. The reactions were heated to 95°C for 15 min; then 95°C for 15 sec, 55°C for 15 sec, and 72°C for 30 sec for 25 cycles; and finally 72°C for 10 min. The first round reaction products were diluted 1 : 100 with water and re-amplified in an identical manner using the HIVin2 and SILKREVla primers. The final products were separated by electrophoresis in agarose, isolated using a using a QIAquick Gel Extraction Kit (Qiagen), and cloned into the pCR4-TOPO vector using a TOPO TA Cloning Kit (Invitrogen Corp.; Carlsbad CA). Dye-terminator sequencing of eight clones was performed using the primers supplied with the kit. One of the sequenced clones was used to create an integration junction standard curve in the TaqMan reaction above.

Preparation of salt-stripped PICs

Salt-stripped PICs were prepared by a modification of previously described methods (Lee and Craigie, Proc. Natl. Acad. Sci. -JS--4 95, 1528-1533, 1998; Chen and Engelman, Proc. Natl. Acad. Sci. USA 95:15270-15274, 1998, each of which is incorporated herein by reference). Five hundred μl of PIC-containing cytoplasmic extract was diluted in an equal volume of Buffer A containing 0.025% digitonin, but without KC1 to reduce the salt concentration to 75 mM. The diluted PICs were incubated at 4°C for 30 min, then pelleted at 8,000 g for 20 min in a refrigerated microcentrifuge. The barely visible pellet was resuspended in 170 μl of Buffer A with 1.2 M KC1 and incubated for 30 min on ice.

The hypertonic PIC solution was loaded onto a Sepharose CL-4B column (2.2 ml bed volume) that had been pre-equilibrated in the same buffer, but not pretreated with BSA. The column was placed in a tube and centrifiiged at 800g for 3 min at 4°C. Approximately 1500 μl was retrieved from each column, then concentrated to about 40 μl by centrifugation at 800g in a Microcon- 100 ultrafiltration unit (Millipore), taking care not to allow the sample to concentrate to dryness. The amount of HIV LTR cDNA was quantified using the TaqMan™ assay so that samples could be adjusted to equal numbers of PICs before placing them in the integration assay. Reconstitution of salt-stripped PICs

Cytoplasmic extracts were prepared from uninfected SupTl cells using the same protocol used to prepare PIC-containing extracts. Five μl of the extract was added to 2 μl of salt-stripped PICs and incubated on ice for 15 min. Five μl of Buffer A without salt was added to bring the KCl concentration to about 262 mM, and the mixture was incubated on ice for another 15 min to allow the cytoplasmic proteins to assemble onto the salt-stripped PICs. Ten μl of the mixture was analyzed in the integration assay.

Modified Real-time PCR

Because the forward primer (SEQ ID NO:l) and probe (SEQ ID NO:3) both bind to the same (bottom; see Figure 7) strand of the 5' LTR and 3' LTR, regardless of whether the LTR has integrated, an arithmetic increase in TaqMan™ signal can be generated from this source alone. When large amounts of HIV cDNA are present, the signal appears as a rising baseline during the TaqMan™ run. The bottom strand of the integration junction is created by the SILKREVla primer (SEQ ID NO:2). Unless this occurs, there is no binding site on the junctional strand for the forward primer, LTRTaq5 (SEQ ID NO.T). Increasing the amount of the "top" strand of the silk sequence (see Figure 7) can inhibit the PCR reaction. This is the reason why the removal of excess target DNA using λ exonuclease is so successful.

Various approaches can be used to alleviate such potential problems. For example, the probe (SEQ ID NO: 3) can be re-designed as the complementary inverse sequence, such that it binds in the reverse direction to the top strand (Figure 7). Such a probe is exemplified by the following sequence:

5'-FAM-CTGACTAAAAGGGTCTGAGGGATCTCTAGTTACCAG-TAMRA-3'

(SEQ ID NO: 17).

As such, the Taq polymerase 5' exonuclease activity generates a fluorescent signal when the reverse primer (SILKREVla; SEQ ID NO:2) is extended, which only can occur on the junctional strand, thereby eliminating the "noise" from unintegrated LTR cDNA. In this assay, it is beneficial to increase the efficiency with which the bottom junctional strand (see Figure 7) is created. This process is dependent on the reverse primer (SILKREVla; SEQ ID NO:2), which has many binding sites on the target DNA, even after λ exonuclease digestion. One way to increase the efficiency of "bottom" strand extension is to add a nucleotide sequence at the end of the target nucleic acid molecule, for example, S32, that can be altered to form an intramolecular "snap back" loop, which can hybridize in the reverse direction and autoprime the formation of the bottom junctional strand.

Another way to increase efficiency of bottom strand extension is to include a reverse primer ("TargetRev") to the reaction, wherein the reverse primer binds to the sequence following the terminal Sty I site of the silk repeating units in the target DNA (see Figure 7). The TargetRev primer need not be designed to amplify the junctional strand, as the SILKREVl a (SEQ ID NO:2) primer serves that purpose efficiently, and the products of a LTRTaq5 (SEQ ID NO:l) and SILKREVla (SEQ ID NO:2) amplification will not have a binding site for the TargetRev primer. However, if the TargetRev primer hybridized at a higher temperature (e.g., greater than 70°C), then cycles of linear preamplification can be included in the TaqMan program (e.g., 95°C for 15 sec, and 70°C for 30 sec). Ten, 20, 30, or 100 such cycles can increase the final TaqMan™ signal by the respective number.

The TargetRev primer can be designed to hybridize only to the target DNA. However, at the lower temperatures used for the TaqMan™ reaction (e.g., 60°C), the primer can have a reduced selectivity of hybridization and combine, for example, with one of the other primers to generate erroneous amplification products that can interfere with the assay. To avoid this, TargetRev could be designed as a step loop, panhandle structure which auto-suppresses intermolecular hybridization at the lower temperature.

A method of PCR suppression also can be used. PCR suppression is based on the finding that DNA strands that ends with a "pan handle" or "hairpin" stem-loop structure do not generally bind a PCR primer (Lauiier et al., Mol. Gen. Mikrobiol. Virusol. 6:38-41, 1994; Lukyanov et al., Anal. Biochem. 229:198-202, 1995; Siebert et al, Nucl. Acids Res. 23:1087-8, 1995; Broude et al., Proc. Natl. Acad. Sci.. USA 98:206-11, 2001; U.S. Pat. Nos. 5,565,340 and 5,759,822, each of which is incorporated herein by reference), can be used. Using this approach, intramolecular hybridization is a first order reaction, whereas intermolecular hybridization is a second order reaction. These principles apply in reverse if a DNA primer is made to contain a stem-loop structure (analogous to a molecular beacon), in which case the specificity of the primer binding is enhanced (Bonnet et al., Proc. Natl. Acad. Sci., USA 96. 11:6171-6, 1999, which is incorporated herein by reference). At temperatures below the melting temperature of the stem-loop structure, the primer is unable to hybridize to the target and, therefore, can be used to achieve a hot start effect during PCR (Kaboev et al., Nucl. Acids Res. 28:E94, 2000, which is incorporated herein by reference). In this application, the stem-loop primer is used at a high temperature to prime linearly amplified copies of the bottom strand of an integration junction, then the temperature is lowered for the annealing/extension stages of the TaqMan™ reaction, under which the stem-loop primer is unable to participate in intermolecular binding and primer extension. This allows the exponential TaqMan™ amplification phase to proceed as expected without adding a second reverse primer to the already optimized TaqMan^T reaction.

The TargetRev primer is designed for PCR suppression by incorporating three sections of sequence: A, B, and C, wherein A and C can hybridize to the target sequence at temperatures less than 65°C, ensuring that there is little if any intermolecular hybridization at the 60°C temperature used in the annealing/extension stages of the TaqMan™ reaction. Alternatively, if A and B together hybridize to the target nucleic acid molecule between the terminal Sty I site and the Bam HI site at the 3' end (see Figure 7) or an analogously located sequence in a different target DNA with a Tm greater than 75°C, linear amplification of the target DNA can occur. Also, from this design, intermolecular hybridization of TargetRev, where A hybridizes to C on a different oligonucleotide and vice versa, is suppressed by the stronger intramolecular hybridization. B. RESULTS

A concatemeric DNA substrate containing 32 head-to-tail monomeric units encoding drag-line silk, a natural protein composed of repeating units (Winkler et al, Int. J. Biol. Macromol. 24, 265-270, 1999, which is incorporated herein by reference), was used to investigate the effect of target DNA length on the efficiency of integration. In order to amplify integration junctions, PCR primers were designed to hybridize to the 3' LTR of HIV-1 (forward primer) and the silk target DNA (reverse primer). The concatemeric arrangement of the target DNA placed a binding site for the reverse primer within a short distance (<183 bp) from the LTR forward primer, thus effectively limiting amplicon size to a length that is optimal for TaqMan™ detection. Although both the 5' and 3' LTRs integrate into a target DNA during concerted integration, only the integration of the 3' LTR was analyzed in these studies. Also, although HIV can integrate into the target DNA in either orientation, the assay only detects integration junctions in one of the two target DNA strands (see Figure 1; shown as the top strand).

Amplicons containing putative integration junctions were isolated by hemi-nested PCR and cloned into a plasmid. Sequencing analysis revealed that the integrations had occurred into various sites of the target DNA, with a possible "hot spot" immediately 5' to the reverse primer. In several cases, the cloned amplicons contained an additional monomeric unit of target DNA on their 3' end, presumably due to the hybridization of the reverse primer to a binding site in the next unit adjacent to the integration site.

The real-time PCR assay was validated using control reactions that lacked either PICs or target DNA. As expected for a 45 cycle real-time PCR, the negative control integration reactions yielded values equal to 45, indicating that no integration junctions were detected. To quantify positive reactions, dilutions of a cloned integration junction were prepared to create the standard curves used to relate the C_t values obtained from an unknown sample to the number of integration events that occurred. When reaction mixtures were diluted prior to detection, the final number of integration events reported was calculated by multiplying by the dilution factor. No correction was performed to account for the presumably equal number of integrations that would be expected to occur into the complementary strand of the target DNA, which was not measured.

When PICs and target DNA were both present in an integration reaction and the λ exonuclease processing step was omitted, a signal corresponding to about 500 to 1,000 integration events was detected (Figure 2). When the reaction mixtures were diluted prior to PCR, the number of integration events detected (corrected for the dilution factor) increased, indicating that the reaction mixtures contain an inhibitor for the PCR detection step. This nonlinear response may explain why there has been no previous report of a quantitative PIC assay based upon the PCR quantification of integration junctions.

An exhaustive analysis revealed that the primary source of the inhibition of detection was carry-over into the PCR detection step of unreacted target DNA, which contains a large number of binding sites for the reverse primer. For example, the addition of target DNA, but not irrelevant plasmid DNA, to a cloned integration junction dramatically inhibited real-time PCR detection. Increasing the concentration of the reverse primer was increased 10-fold (to 9 μM final concentration) completely reversed the inhibitory effects of target DNA on the detection of 10 and 100 copies of a cloned integration junction. However, for the integration junctions generated by PICs in the reaction mixtures, PCR amplification depends upon the reverse primer creating the complementary strand to which the forward primer binds (whereas this strand preexists when a cloned integration junction is used). In this case, a simple increase in the reverse primer concentration was not sufficient to eliminate the nonlinearity of the assay.

A close examination of the amplification plots revealed an additional problem caused by the carry-over of target DNA into the TaqMan™ reaction. More specifically, the slope of the amplification plot was markedly less than that of the cloned integration junction that was used as a standard. In effect, the target DNA prevented the amount of product from doubling with each cycle of PCR. The atypical shape of the amplification plots from the samples containing target DNA reduced the sensitivity of the TaqMan™ analysis by raising the apparent , and also precluded the possibility of converting the assay to an end-point detection format.

Two strategies were identified that removed the nonlinearity of the assay by reducing or eliminating the carry-over of target DNA into the PCR detection step. The first strategy was to dilute the integration reaction mixtures prior to PCR detection. However, dilution of the reaction mixtures also dilutes the integration junctions that are to be detected real-time PCR. As a result, diluting the reaction mixtures more than about 100-fold prior to TaqMan™ detection generally resulted in C_t values outside of the dynamic range of the assay.

The second strategy to selectively remove the target DNA utilized λ exonuclease, which degrades double stranded DNA having a 5 '-phosphorylated overhang. This approach did not affect the sensitivity of the TaqMan™ assay. An examination of the λ exonuclease-treated reactions by polyacrylamide gel electrophoresis and SYBR Gold nucleic acid staining demonstrated that all visible DNA in the reactions was removed. Furthermore, the λ exonuclease did not affect the integration junctions, perhaps due to protection of the junctions by PIC proteins bound to the DNA (Miller et al., supra, 1997). The PIC proteins may be part of the intasome complex that has been detected on the ends of integrated MMLV LTR DNA (Wei et al, EMBO J. 16:7511-7520, 1997). Pre-treatment of an integration reaction with λ exonuclease prior to detection consistently resulted in a 4-fold to 10-fold increase in detection sensitivity (Figure 2). The amplification plots of the λ exonuclease-treated samples showed a normal slope in parallel to the cloned integration junction standards, indicating that the method can be used in a simplified end-point detection format.

The effect of target DNA length on the efficiency of PIC integration in vitro was examined by using target DNA containing one (SI) or 32 (S32) monomeric units of the drag-silk coding sequence (Figure 3). The results indicate that, 1) for a given number of monomeric units of target DNA present in the integration reaction, the concatemerization of those units into a longer molecule resulted in a 2-fold to greater than 10-fold increase in the integration efficiency; and 2) integration efficiency increases with an increase in the amount of target DNA molecules (in terms of monomeric units). These results also indicate that the number of integration events are maximized by keeping a relatively high amount of target DNA in the integration reaction. The linearity of the assay for PICs was greatest at a concentration of about 9.27 x 10¹⁰ monomers or less/15 μl reaction, a concentration that can be obtained using the 32-mer target DNA.

The ability of the integration assay system using long target DNA concatemers to measure integration competent PICs was validated by examining serially diluted PICs. The number of integration events was quantified in undiluted integration reactions using 32-mer concatemeric target DNA and λ exonuclease treatment. The number of integration events decreased proportionally to PIC dilution (Figure 4). For the PIC extract tested, dilutions greater 9-fold produced C_t values that were outside of the linear range of the assay. In other experiments, in which the target DNA concentration was reduced, less inhibition of the real-time PCR detection step resulted. In the latter experiments, integration events were detectable in PIC extracts diluted 27-fold, even though the absolute number of integration events at each dilution was lower. These results demonstrate that the PIC assay system using long concatemeric target DNA can reliably quantify the number of integration competent PICs in a sample. Furthermore, the integration assay is so sensitive that a single 50 ml culture of infected SupTl cells generates enough PICs for over 10,000 assays. As such, the assay can utilize authentic HIV-1 PICs as the basis of a high throughput drug discovery program. The assay also can be used to guide the purification of PICs by ultracentrifugation in a sucrose gradient.

The effect of compounds known to inhibit HIV-1 integration in vitro was examined using the integration assay. Initially, the stability of PIC extracts at 37°C was examined. PIC-containing cytoplasmic extracts were stable at 37°C for about 20 min, but became unable to integrate into target DNA by one hour (Figure 5, inset). The effect of two known inhibitors of HIV-1 integrase, purpurin and quinalizarin, then were examined. Both integrase inhibitors clearly inhibited integration (Figure 5). These results demonstrate that the present integration assay using concatemeric target DNA can be used to identify integrase-directed PIC inhibitors (IPIs).

The ability of the integration assay to identify viral or cellular factors involved in the integration reaction also was examined. Factors required for integration were removed from the PICs under conditions of high salt (Lee and Craigie, Proc. Natl. Acad. Sci.. USA 91 :9823-9827, 1994, which is incorporated herein by reference). The "salt-stripped" HIV-1 PICs had no detectable integration competence using the PIC integration assay system with long, concatemeric target DNA. However, integration competence was restored when cytoplasmic extracts of uninfected SupTl cells were added to the salt-stripped PICs (Figure 6). These results demonstrate that the PIC integration assay system can be used to identify host cell factors that contribute to PIC activity. Furthermore, this aspect of the assay can be used to identify antiretro viral compounds that can inhibit the function of PIC-activating host cell factors, i.e., non-integrase-directed PIC inhibitors (NIPIs), which can prevent the assembly of active PICs or disrupt the activity of a PIC. Although assays such as the strand transfer assay can be used to identify integrase-directed PIC inhibitors (IPIs), the present assay appears to be the first assay that can be adapted to detect NIPIs in a high-throughput format.

EXAMPLE 2

IDENTIFICATION AND CHARACTERIZATION OF

PRO-PREINTEGRATION COMPLEXES

This example demonstrates that pro-preintegration complexes (pro-PICs) are present in retrovirus-infected resting cells and that such pro-PICs can be rendered integration competent by factors present in activated cells.

A. METHODS Peripheral blood lymphocytes

Heparinized blood was collected from healthy, HIV-seronegative donors. Peripheral blood mononuclear cells were prepared by centrifugation over ficoll- Hypaque using standard methods, and peripheral blood lymphocytes (PBLs) were isolated after removing monocytes by adherence to plastic (Coligan et al, supra, 2001). PBLs were stimulated to divide by treatment with phytohemagglutinin (PHA) (3 μg/ml) and IL-2 (30 U/ml) for 48 to 72 In- in RPMI 1640 media containing 10% human AB serum. Alternatively, PBLs in RPMI 1640 containing 10% human AB serum were stimulated for various time intervals by culturing them on plates coated with anti-CD3 (Becton Dickenson, Inc.) and anti-CD28 (Pharmingen Inc.) antibodies (Coligan et al, supra, 2001).

Lentiviral vector infection and preparation of cytoplasmic extracts To test the ability of retroviruses to infect resting and activated cells, a VSV-G pseudotyped lentiviral vector system was used (Chinnasamy et al., supra, 2000) based on the constructs described by Naldini et al. (supra, 1996; U.S. Pat. No. 6,218,181). Using this system, plasmids encoding VSV-G envelope protein, HIV-1 structural and accessory proteins, and an expression cassette for green fluorescent protein were transfected into 293T cells. The virions released by these transfected cells were concentrated by ultracentrifugation and titered in MAGI cells. As required, frozen stocks of virus were thawed and used to infect PBLs at a multiplicity of infection (MOI) of 10, as described (Chinnasamy et al., supra, 2000). At various times following lentiviral vector infection, the cells were washed and lysed with digitonin (Farnet and Haseltine, supra, 1990; Miller et al, supra, 1997, each of which is incorporated herein by reference). Nuclei and cell debris were removed by centrifugation, and 40% sucrose was added to the PIC-containing cytoplasmic extracts to a final concentration of 8%. The extracts were then aliquoted, flash frozen with liquid nitrogen, and stored at -70 °C (Farnet and Haseltine, supra, 1990; Miller et al., supra, 1997). These cytoplasmic extracts contained approximately 0.8 to 2 mg/ml of total protein, as measured using the BCA reagent (Pierce Chemical, Rockford, IL), and 0.5-30 x 10⁸/ml copies of HIV-1 cDNA as quantified by TaqMan real-time PCR using LTR-specifϊc primers (Rossio et al., supra, 1998).

B. RESULTS

The VSV-G pseudotyped lentiviral vector system provides an efficient way to generate large amounts of viral cDNA and PICs for analysis. Accordingly, peripheral blood mononuclear cells were depleted of monocytes by plastic adherence. The remaining peripheral blood lymphocytes (PBLs) were cultured for 48 hr in medium, alone, or in medium containing PHA and IL-2. The cells were transduced with the VSV-G-pseudotyped lentiviral vector at a multiplicity of infection (MOI) of 10. PIC-containing cytoplasmic extracts were prepared 6 hr later by lysis with digitonin, followed by centrifugation steps to remove nuclei and cellular debris.

TaqMan™ real-time PCR of early and late reverse transcription products using primers for the LTR and LTR-gag regions of cDNA (Rossio et al., supra, 1998) indicated that there were comparable amounts of partially and fully reverse transcribed cDNA in the cytoplasmic extracts from both resting and activating PBLs. However, there were no detectable integration-competent PICs in the cytoplasmic extracts from resting PBLs. In contrast, integration-competent PICs were readily detected in the cytoplasmic extracts from PHA/IL-2-activated PBLs.

Resting T lymphocytes infected with HIV-1 in vitro or in vivo can be stimulated many days after infection, resulting in integration and the production of new virions. To test the sequence of infection followed by stimulation, resting PBLs were infected using the VSV-G-pseudotyped lentiviral vector, washed, cultured in media for 24 hr, then transferred to either control plates (resting PBLs) or plates coated with anti-CD3 and anti-CD28 antibodies (CD3/CD28 PBLs). PIC-containing cytoplasmic extracts were prepared at 6, 24, and 48 hr after stimulation. As a positive control, cytoplasmic extracts were prepared from SupTl cells acutely infected by HIV-1 LAi (LAV-1) as described above.

TaqMan™ real-time PCR of early and late reverse-transcription products (LTR and LTR-gαg cDNAs) revealed about 10-fold more early and late reverse- transcribed cDNAs in the CD3/CD28-stimulated PBLs compared to the resting PBLs, which is compatible with a previous report (Zack et al, J. Nirol. 66:1717-1725, 1992). Consequently, all samples were adjusted to contain the same amount of LTR cDΝA and then assayed for integration-competent PICs. No integration competent PICs were detected in resting PBLs infected 24 hr previously, whereas, after the same cells were stimulated, integration competent PICs were detected. The integration competent PICs appeared by 6 hr after CD3/CD28 stimulation and were maximal at 24 hr after stimulation. No integration-competent PICs were detected at 48 hr after transduction, when the level of LTR cDNA had begun to fall, presumably because the PICs had undergone import into the nuclei of these activated T cells (Polacino et al., J. Exp. Med. 182:617-621, 1995) and, therefore, were no longer present in the cytoplasmic extract being examined.

These results indicate that integration competent PICs do not form in cultured human lymphocytes in the absence of cellular activation. Instead, there is a block at a stage after reverse transcription, resulting in the formation of a "pro-preintegration complex" ("pro-PIC"). The results further indicate that host cell factors that are induced due to T cell activation are required for the conversion of pro-PICs to integration-competent PICs. Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of detecting integration of a polynucleotide into a target nucleic acid molecule, the method comprising: a) contacting a sample comprising the polynucleotide and the target nucleic acid molecule, under conditions that allow integration of a polynucleotide into the target nucleic acid molecule; and b) detecting covalent linkage of the polynucleotide and the target nucleic acid molecule, thereby detecting integration of the polynucleotide into the target nucleic acid molecule.

2. The method of claim 1, wherein the polynucleotide comprises a virus polynucleotide.

3. The method of claim 2, wherein the virus is a retrovirus.

4. The method of claim 3, wherein the retrovirus is a lentivirus.

5. The method of claim 4, wherein the lentivirus is a human immunodeficiency virus (HIN).

6. The method of claim 5, wherein the HIV is HIV-1 or HIV-2.

7. The method of claim 1, wherein the polynucleotide comprises at least one repeat sequence of a retrovirus long terminal repeat.

8. The method of claim 1, wherein the polynucleotide comprises a retrovirus long terminal repeat.

9. The method of claim 1 , wherein the polynucleotide is a transposable element.

10. The method of claim 9, wherein the transposable element is a yeast transposon or a plant transposon.

11. The method of claim 1 , wherein the polynucleotide comprises a retroelement of a eukaryotic genome.

12. The method of claim 11 , wherein the eukaryotic genome is a mammalian genome.

13. The method of claim 1 , wherein the polynucleotide is associated with an integrase.

14. The method of claim 1, wherein the polynucleotide is associated with a pre-integration complex.

15. The method of claim 1, wherein target nucleic acid molecule comprises a concatemer of target nucleic acid molecules.

16. The method of claim 1, wherein target nucleic acid molecule comprises a nucleosome.

17. The method of claim 1, further comprising, after a) and before b), contacting the sample with an exonuclease, under conditions sufficient for the exonuclease to substantially degrade unreacted target nucleic acid molecules; diluting the sample; or contacting the sample with the exonuclease and diluting the sample.

18. The method of claim 1, wherein detecting covalent linkage of the polynucleotide and the target nucleic acid molecule comprises: contacting the sample with an exonuclease having 5' exonuclease activity; thereafter contacting the sample with an primer that selectively hybridizes at or near the 3' terminus of the target nucleic acid molecule, under conditions that allow primer extension from the oligonucleotide primer; and detecting generation of the primer extension product.

19. The method of claim 18, wherein detecting generation of the primer extension product comprises: contacting the sample with an amplification primer pair, comprising a first primer that selectively hybridizes to a nucleotide sequence of the polynucleotide, and a second primer that selectively hybridizes to a nucleotide sequence of the primer extension product, under conditions that allow generation of an amplification product from the amplification primer pair, wherein the conditions that allow generation of the amplification comprise conditions below the melting temperature of a secondary structure formed by the oligonucleotide primer, thereby preventing the oligonucleotide primer from selectively hybridizing at or near the 3' terminus of the target nucleic acid molecule; and detecting generation of the amplification product.

20. The method of claim 1, wherein detecting covalent linkage of the polynucleotide and the target nucleic acid molecule comprises: contacting the sample with a first amplification primer pair, comprising a first primer that selectively hybridizes to a nucleotide sequence of the polynucleotide, and a second primer that selectively hybridizes to a nucleotide sequence of the target nucleic acid molecule, under conditions that allow generation of a first amplification product from the primer pair; and detecting generation of an amplification product.

21. The method of claim 20, wherein detecting generation of the amplification product comprises detecting the first amplification product.

22. The method of claim 20, further comprising contacting the sample with a detector oligonucleotide, which can selectively hybridize to a nucleotide sequence of the first amplification product; and detecting selective hybridization of the detector oligonucleotide to the first amplification product.

23. The method of claim 20, further comprising contacting the sample with a bilabeled oligonucleotide probe, which comprises a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the first amplification product; and detecting fluorescence due to the fluorescent moiety.

24. The method of claim 20, wherein detecting generation of an amplification product comprises: contacting the sample with at least a first primer of a second amplification primer pair, wherein the first primer of the second amplification primer pair can selectively hybridize to a nucleotide sequence of the first amplification product, under conditions that, in the presence of a second primer of the second amplification primer pair, allow generation of a second amplification product; and generating a second amplification product.

25. The method of claim 24, wherein the first primer or second primer of the first amplification primer pair is the second primer of the second amplification primer pair.

26. The method of claim 24, further comprising contacting the sample with the second primer of the second amplification primer pair.

27. A method of detecting integration of a polynucleotide into a target nucleic acid molecule, the method comprising: a) contacting a sample with a pre-integration complex (PIC), which comprises the polynucleotide and an integrase, and the target nucleic acid molecule, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule; b) thereafter decreasing the effective concentration of target nucleic acid molecules in the sample; c) further contacting the sample with a first amplification primer pair, under conditions that allow generation of a first amplification product from an integration product of the polynucleotide of the PIC and the target nucleic acid molecule, wherein the first amplification primer pair comprises a first primer that selectively hybridizes to a nucleotide sequence of the polynucleotide of the PIC, and a second primer that selectively hybridizes to a nucleotide sequence of the target nucleic acid molecule; and d) detecting generation of an amplification product, thereby detecting integration of the polynucleotide into the target nucleic acid molecule.

28. The method of claim 27, wherein decreasing the effective concentration of target nucleic acid molecules in the sample comprises: diluting the sample; contacting the sample with an exonuclease, which degrades unreacted target nucleic acid molecules; or a combination thereof.

29. The method of claim 27, wherein, in step c), the sample is further contacted with a bilabeled oligonucleotide probe, comprising a fluorescent moiety and a fluorescent quencher, wherein the oligonucleotide probe selectively hybridizes to a nucleotide sequence of the polynucleotide downstream from the first amplification primer, or a nucleotide sequence of the target nucleic acid molecule downstream from the second amplification primer; and wherein detecting generation of the amplification product comprises detecting fluorescence due to the fluorescent moiety.

30. The method of claim 27, wherein detecting generation of an amplification product comprises: contacting the first amplification with at least a first primer of a second amplification primer pair, under conditions that, in the presence of a second primer of the second amplification primer pair, allow generation of a second amplification product, wherein the first primer of the second amplification primer pair selectively hybridizes to a nucleotide sequence of the first amplification product; and detecting generation of the second amplification product.

31. A method of identifying an agent that modulates integration of a polynucleotide into a target nucleic acid molecule, the method comprising: a) contacting in solution the polynucleotide, the target nucleic acid molecule, and a test agent, under conditions that allow integration of the polynucleotide into the target nucleic acid molecule; and b) detecting a change in the number of integration events of the polynucleotide and the target nucleic acid molecule in the presence of the test agent as compared to the number of integration events in the absence of the test agent, thereby detecting an agent that modulates integration of the polynucleotide into the target nucleic acid molecule.

32. The method of claim 31 , wherein the agent reduces or inhibits the number of integration events.

33. The method of claim 31 , wherein the agent increases the number of integration events.

34. The method of claim 31, wherein the number of integration events is detected after a predetermined time of contacting.

35. The method of claim 31, wherein the test molecule is a peptide, a 5 peptidomimetic, or a polynucleotide.

36. The method of claim 31, wherein the test agent comprises one of a library of test agents.

10 37. The method of claim 31, which is performed in a high throughput format.

38. The method of claim 31, wherein the polynucleotide comprises a pro-preintegration complex, the method comprising identifying an agent that renders a pro-preintegration complex integration competent.

15

39. The method of claim 38, wherein the pro-preintegration complex comprises a retroviral vector genome.

40. A method of identifying a factor that mediates integration of a 20 polynucleotide into a target nucleic acid molecule, the method comprising:

A a) contacting in solution the polynucleotide, the target nucleic acid molecule, and a test factor, under conditions that allow integration of the. polynucleotide into the target nucleic acid molecule; b) detecting integration of the polynucleotide into the target nucleic

25 acid molecule, thereby identifying a factor involved in integration of the polynucleotide into the target nucleic acid molecule.

41. The method of claim 40, wherein the factor that mediates integration comprises a cellular factor.

30

42. The method of claim 41, further comprising isolating the cellular factor.

43. An isolated cellular factor involved in integration of a polynucleotide into a target nucleic acid, wherein the factor is identified by the method of claim 40.

44. The method of claim 40, wherein the test factor comprises a synthetic molecule.

45. The method of claim 40, wherein the test factor comprises one of a library of test factors.

46. The method of claim 40, wherein the test factor is a peptide, a peptidomimetic, or a polynucleotide.

47. The method of claim 40, wherein the test factor is a cellular factor.

48. The method of claim 47, wherein the cellular factor is obtained from a virally infected cell.

49. The method of claim 40, wherein the polynucleotide comprises a viral polynucleotide.

50. The method of claim 49, wherein the viral polynucleotide comprises a complex with an integrase.

51. The method of claim 49, wherein the viral polynucleotide is obtained from a cell infected with virus comprising the viral polynucleotide.

52. The method of claim 51, wherein the viral polynucleotide comprises a pre-integration complex.

53. The method of claim 51, wherein the viral polynucleotide comprises a salt-stripped pre-integration complex.

54. The method of claim 51 , wherein the viral polynucleotide comprises a pro-pre-integration complex.

55. A method of generating a linear amplification product of a selected strand of a nucleic acid molecule, the method comprising contacting a sample comprising the nucleic acid molecule with at least one oligonucleotide primer that selectively hybridizes to the selected strand, under conditions that allow generation of a linear amplification product comprising the oligonucleotide primer, wherein the oligonucleotide primer can form a secondary structure, which prevents the primer from selectively hybridizing to the selected strand, and wherein the conditions that allow generation of the linear amplification product comprise conditions above the melting temperature of the secondary structure,

* thereby generating a linear amplification product of the selected strand.

56. The method of claim 55, further comprising contacting the linear amplification product with an amplification primer pair, comprising a first primer that selectively hybridizes to a nucleotide sequence of the selected strand of the nucleic acid molecule, and a second primer that selectively hybridizes to a nucleotide sequence of the linear amplification product, under conditions that allow generation of an amplification product from the amplification primer pair, wherein the conditions that allow generation of the amplification product comprise conditions below the melting temperature of a secondary structure formed by the oligonucleotide primer, thereby preventing the oligonucleotide primer from selectively hybridizing to the selected strand, thereby obtaining an amplification product comprising a nucleotide sequence of the selected strand.

57. The method of claim 55, wherein the oligonucleotide primer selectively hybridizes at or near a 3' terminus of the nucleic acid molecule.

58. The method of claim 55, wherein the nucleic acid molecule is a single stranded nucleic acid molecule, said single strand comprising the selected strand.

59. The method of claim 58, wherein the single stranded nucleic acid molecule is a deoxyribonucleic acid strand complementary to an mRNA.

60. The method of claim 55, wherein the nucleic acid molecule is a double stranded nucleic acid molecule, comprising a first strand and a second strand.

61. The method of claim 60, wherein the first strand comprises a nick, wherein the second strand is the selected strand, and wherein the linear amplification product spans the nick.

62. The method of claim 61, wherein the selected strand comprises an integration j unction.

63. The method of claim 55, further comprising detecting generation of the linear amplification product, said detecting comprising a) contacting the sample with a bilabeled oligonucleotide probe, which comprises a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the selected strand that is in a 3' position with respect to the oligonucleotide primer; and b) detecting fluorescence due to the fluorescent moiety.

64. The method of claim 56, further comprising detecting generation of the amplification product, said detecting comprising a) contacting the sample with a bilabeled oligonucleotide probe, which comprises a fluorescent moiety and a fluorescence quencher moiety, wherein the bilabeled oligonucleotide probe can selectively hybridize to a nucleotide sequence of the amplification product; and b) detecting fluorescence due to the fluorescent moiety.

65. The method of claim 55, wherein the sample comprising the nucleic acid molecule is a cell sample.

66. The method of claim 65, wherein the cell sample is obtained from a subject.

67. The method of claim 66, wherein the cell sample is obtained using a biopsy procedure.

68. The method of claim 66, wherein the subject is a vertebrate.

69. The method of claim 66, wherein the subject is a human.

70. The method of claim 55, wherein the oligonucleotide primer selectively hybridizes to a conserved nucleotide sequence, which is common to a population of related but different nucleic acid molecules.

71. The method of claim 70, wherein the conserved sequence encodes a polypeptide domain or a peptide portion thereof.

72. The method of 55, further comprising contacting the linear amplification product with a hybridization probe, which can selectively hybridize to a nucleotide sequence of the linear amplification product.

73. The method of claim 72, wherein the hybridization probe is attached to a solid support.

74. The method of claim 55, further comprising isolating the linear amplification product.

75. The method of claim 72, wherein the hybridization probe comprises a plurality of different hybridization probes.

76. The method of claim 75, wherein the solid support is a microchip, a glass slide, or a bead, and wherein the hybridization probes of the plurality are in an array.

77. A kit, comprising: a target nucleic acid molecule; at least one primer of a first amplification primer pair; wherein said primer can selectively hybridize to a nucleotide sequence of the target nucleic acid molecule; and an exonuclease.

78. The kit of claim 77, wherein the target nucleic acid is about 10 to 1000 nucleotides in length.

79. The kit of claim 77, wherein the target nucleic acid is about 50 to 500 nucleotides in length.

80. The kit of claim 77, wherein the target nucleic acid is about 100 to

250 nucleotides in length.

81. The kit of claim 77, wherein the target nucleic acid molecule comprises a concatemer of target nucleic acid molecules.

82. The kit of claim 81, wherein the concatemer comprises about 2 to 1000 target nucleic acid molecules.

83. The kit of claim 81, wherein the concatemer comprises about 10 to 250 target nucleic acid molecules.

84. The kit of claim 83, wherein the concatemer comprises about 25 to 100 target nucleic acid molecules.

85. The kit of claim 77, further comprising a polynucleotide that can integrate into the target nucleic acid molecule.

86. The kit of claim 85, further comprising a second primer of the amplification primer pair, wherein the second primer selectively hybridizes to a nucleotide sequence of the polynucleotide that can integrate into the target nucleic acid molecule.

87. The kit of claim 85, further comprising an integrase that can mediate integration of the polynucleotide.

88. The kit of claim 77, further comprising a detector oligonucleotide, which can selectively hybridize to an amplification product generated by the amplification primer pair.

89. The kit of claim 77, further comprising at least one primer of a second amplification primer pair, wherein said primer can selectively hybridize to a nucleotide sequence of an amplification product generated using the first amplification primer pair.

90. A kit, which contains, in a single tube, an integrating polynucleotide, a target nucleic acid molecule, and a buffer that provides conditions sufficient for integration of the integrating polynucleotide into the target nucleic acid molecule.

91. The kit of claim 90, wherein the single tube further contains an amount of λ exonuclease sufficient to degrade unreacted target nucleic acid molecules following integration of the integrating polynucleotide into the target nucleic acid molecule.

92. The kit of claim 91, wherein the single tube further contains at least one primer that can selectively hybridize to the target nucleic acid molecule, and a DNA polymerase.

93. The kit of claim 92, wherein the single tube further contains a detector oligonucleotide.

94. The kit of claim 91, wherein the single tube further contains at least one primer that can selectively hybridize to the integrating polynucleotide, and a DNA polymerase.

95. The kit of claim 94, wherein the single tube further contains a detector oligonucleotide.