WO1997025444A1

WO1997025444A1 - Method of monitoring pharmacokinetics of oligonucleotide pharmaceuticals

Info

Publication number: WO1997025444A1
Application number: PCT/US1996/020266
Authority: WO
Inventors: Aharon S. Cohen; Andre J. Bourque; Bing Wang; Alexei Belenky
Original assignee: Hybridon, Inc.
Priority date: 1996-01-16
Filing date: 1996-12-18
Publication date: 1997-07-17
Also published as: AU1428097A

Abstract

The invention provides a method for measuring the amounts of oligonucleotides, particularly applicable to monitoring the pharmacokinetics of antisense oligonucleotide pharmaceuticals, which permits resolution of homologous oligonucleotides differing by a single terminal nucleotide. The method consists in the ligation of the target oligonucleotide to a (labeled) primer with the help of a bridging oligonucleotide hybridizing to the ends of both target and primer.

Description

METHOD OF MONITORING PHARMACOKINETICS OF OLIGONUCLEOTIDE PHARMACEUTICALS

The present invention relates to a method of monitoring the in vivo concentrations of oligonucleotide pharmaceuticals and the metabolites of oligonucleotide pharmaceuticals.

BACKGROUND OF THE INVENTION

Antisense oligonucleotide technology has enabled exciting new potential therapies to be developed for many diseases, including pathogenic infections, cancer, and inherited conditions. The field has progressed enormously over the past decade, and currently numerous clinical trials are in progress or are proposed for a variety of antisense oligonucleotide pharmaceuticals. Antisense oligonucleotide pharmaceuticals act by binding to nucleic acid sequences characteristic of the disease or condition, by Watson-Crick or Hoogstein base-pairing, thereby inhibiting transcription or translation of a gene. Antisense oligonucleotide pharmaceuticals may be designed to inhibit transcription or translation of any single gene within an organism's genome. Oligonucleotides containing only phosphodiester internucleotide linkages are known to degraded rapidly both in vitro and in vivo, by virtue of the action of nucleases ubiquitously present in biological fluids. Antisense oligonucleotide pharmaceuticals have therefore been developed which are chemically modified to increase nuclease resistance. Antisense oligonucleotide pharmaceuticals may also contain chemical modifications which enhance uptake into cells, or which minimize side effects, or which act in other ways to optimize the efficacy of the pharmaceutical. The most widely studied antisense oligonucleotide pharmaceuticals, the phosphorothioate oligonucleotide pharmaceuticals, contain sulfur substitutions at the non-bridging oxygens of internucleotide linkages. Phosphorothioate oligonucleotide pharmaceuticals are extremely stable to nuclease degradation in a variety of in vitro systems, e.g., in cell culture medium, in cells and cell extracts, in serum and plasma, in various tissues, and in urine. Studies of pharmacokinetics, i.e., the quantitative guide to the route that a pharmaceutical takes in vivo, from entry into the body, distribution and metabolism within the body, and excretion from the body, are important for preclinical and clinical testing, regulatory approval, and for monitoring of patients receiving the pharmaceutical after regulatory approval. As with other pharmaceuticals, the pharmacokinetics of antisense oligonucleotide pharmaceuticals must be determined both for regulatory approval and during clinical use, and such determinations must be made in efficient and cost-effective ways.

The pharmacokinetics of antisense oligonucleotide pharmaceuticals has traditionally been monitored using known methods for nucleic acid analysis such as radioactive labeling of 3' or 5' phosphate groups with ³²P or ³⁵S, fluorescent labeling, and the like. Although uniform labeling of oligonucleotides with radioactive isotopes is possible, when ³²P or "S is used there are limitations on the number of radioactive atoms which can be present on the oligonucleotide, and uniform labeling with less energetic isotopes is expensive and time consuming. Other monitoring methods such as polyacrylamide gel electrophoresis, HPLC, mass spectrometry, and ultraviolet absorption have also been used to measure oligonucleotide pharmaceuticals in biological samples. PCT/US95/01048 discloses a method of detecting oligonucleotides in biological fluids which employs hybridization of a fluorescently labeled primer oligonucleotide and a helper oligonucleotide to a target oligonucleotide pharmaceutical, ligation of hybridized primer and helper, separation of the ligation product from the target oligonucleotide, and detection of the fluorescent ligation product.

Early preclinical studies in animals have indicated that ³⁵S-labeled phosphorothioate obgonucleotides of varying lengths (20 to 27 nucleotides) and differing base compositions remain intact and have half-lives of 40 to 72 hours in plasma, and that only a small amount of metabolic degradation was found in urine (Iversen, P. in Antisense Research and Application, T.S. Crooke, et al., eds. CRC Press (Boca Raton, FL, 1993) 461; Iversen, P., et al., Antisense Res. Dev. (1994) 4, 43).

As their clinical use becomes more common, it will be necessary to provide accurate and reliable methods for monitoring the in vivo pharmokinetics of antisense oligonucleotide pharmaceuticals. SUMMARY OF THE INVENTION

The present inventors have discovered that the pharmacokinetics of antisense oligonucleotide pharmaceuticals differs from the in vitro behavior of these compounds. This exonuclease activity predominantly degrades antisense oligonucleotides at their 3' termini, in a stepwise fashion. Chronic administration of antisense oligonucleotide pharmaceuticals to mammals results in biological samples containing a mixture of oligonucleotide fragments digested at 3' and 5' termini. The present inventors have devised a method of resolving these oligonucleotide fragments in such biological samples, thus for the first time permitting precise determinations of pharmacokinetics of anusense oligonucleotide pharmaceuticals. In the present method, the primer and target oligonucleotides are hybπdized to an oligonucleotide bridge, and the hybridized primer and target oligonucleotides are ligated to each other. The amount of ligation product is then measured.

In one embodiment, the invention provides an assay comprising: a) providing a sample containing an unknown quantity of a phosphorylated oligonucleotide target having a known sequence, including a predetermined 5' sequence; b) providing an oligonucleotide bridge having a 5' terminal sequence complementary to the predetermined 5' sequence and a predetermined 3' bridge sequence; c) providing an oligonucleotide primer having a 3' terminal sequence complementary to the bridge sequence; d) combining the sample, the oligonucleotide bπdge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; e) ligating the hybridized target and the primer to create a ligation product; and f) measuring the amount of ligation product, wherein the amount of ligaUon product is indicative of the quantity of target in the sample. In another embodiment, the invention provides a method of quantitating a 5'-to-3' linked oligonucleotide target in a biological sample which comprises: a) partially purifying the sample; b) phosphorylating the target at a 5'-terminal nucleotide; c) combining the phosphorylated target with i) an oligonucleotide bridge comprising at least eight 3'-to-5' linked nucleotides, at least four 5'-nucleotides of the bridge having a sequence complementary to at least four 5'-nucleotides of the target; and ii) an oligonucleotide primer having a 3' sequence complementary to a

3'-terminal portion of the bridge; d) incubating the target, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; e) ligating the hybridized target and the primer to create a ligation product; and f) measuring the amount of ligation product, wherein the amount of ligation product is indicative of the quantity of target in the sample.

In another embodiment, the invention provides a method of resolving a mixture of homologous 5' terminal phosphate-containing oligonucleotide species comprising the steps of a) combining the mixture with

(i) an oligonucleotide bridge having a predetermined 3' bridge sequence and a 5' terminal sequence complementary to a region of homology shared by the species; and

(ii) a labeled oligonucleotide primer having a 3' terminal sequence complementary to the bridge sequence; b) incubating the mixture, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; c) ligating the hybridized species and the primer to create a plurality of ligation products; and d) separating the ligation products from the bridge and from each other. In another embodiment, the invention provides an assay comprising: a) providing a sample containing an unknown quantity of an oligonucleotide target having a known sequence, including a predetermined 3' sequence; b) providing an oligonucleotide bridge having a 3' terminal sequence complementary to the predetermined 3' sequence and a predetermined 5' bridge sequence; c) providing an oligonucleotide primer having a 5' phosphate and a 5' terminal sequence complementary to the bridge sequence; d) combining the sample, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; e) ligating the hybridized target and the primer to create a ligation product; and f) measuring the amount of ligation product, wherein the amount of ligation product is indicative of the quantity of target in the sample. In another embodiment, the invention provides a method of quantitating a 5'-to-3' linked oligonucleotide target in a biological sample which comprises: a) partially purifying the sample; b) combining the target with i) an oligonucleotide bridge comprising at least eight 3'-to-5' linked nucleotides, at least four 3'-nucleotides of the bridge having a sequence complementary to at least four 3'-nucleotides of the target; and ii) an oligonucleotide primer having a 5' phosphate and a 5' sequence complementary to a 5'-terminal portion of the bridge; c) incubating the target, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; d) ligating the hybridized target and the primer to create a ligation product; and e) measuring the amount of ligation product, wherein the amount of ligation product is indicative of the quantity of target in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings in which: Figure IA shows the differences in resolution of antisense oligonucleotide pharmaceutical fragments in a blood sample taken from a monkey two hours after infusion of the pharmaceutical, using ion exchange-high performance liquid chromatography.

Figure IB shows the differences in resolution of antisense oligonucleotide pharmaceutical fragments in a blood sample taken from a monkey two hours after infusion of the pharmaceutical using capillary gel electrophoresis.

Figure 2 is a schematic representation of the relationship between the oligonucleotide target pharmaceutical, the oligonucleotide bridge, and the oligonucleotide primer of the method of the present invention.

Figure 3 is a schematic diagram of a capillary gel electrophoresisΛaser-induced fluorescence apparatus suitable for use in the method of the invention.

Figure 4 is an electropherogram of ligation products generated using the method of the present invention as embodied using the apparatus of Figure 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. The issued U.S. patents, allowed applications, and references cited herein are hereby incoφorated by reference. The present invention provides a method of quantitating oligonucleotides, and in particular, oligonucleotide pharmaceuticals, in a biological sample.

Figure 1 is a comparison of techniques used for analysis of aliquots of a single blood sample from a monkey which had been infused with 10 mg kg of a phosphorothioate oligonucleotide pharmaceutical having the sequence set forth in SEQ ID NO:l. The blood sample was harvested and processed as described in Example 1.

The aliquot of Figure IA was subjected to the ion exchange high performance liquid chromatography procedure described in Example 1, and peaks were detected by ultraviolet absorption. The ion exchange-high performance liquid chromatography peak of Figure IA was collected, concentrated, desalted using drop dialysis and subjected to capillary gel electrophoresis. Figure IB shows that the ion exchange-high performance liquid chromatography peak contained at least seven major oligonucleotide components which co-eluted on ion exchange-high performance liquid chromatography but which were resolvable on capillary gel electrophoresis.

Phosphorothioate oligonucleotide pharmaceuticals were found to be degraded in vivo, one nucleotide at a time, initially from the 3' terminus of the oligonucleotide, and over time, from both 3' and 5' termini, with 3' degradation being prevalent. These results contrasted sharply with prior in vivo studies of the pharmacokinetics of phosphorothioate oligonucleotide pharmaceuticals, and indicated that more precise methods are necessary to monitor pharmacokinetics of antisense oligonucleotide pharmaceuticals. The method of the present invention was devised to provide such precision. In accordance with the method of the invention, a biological sample is monitored or assayed for the presence of an oligonucleotide pharmaceutical and its degradation products. Any biological sample may be monitored using the method of the invention. As defined herein, a biological sample may be serum, plasma, whole blood, urine, saliva, sputum, milk, lymphatic fluid, lacrimal secretions, cerebrospinal fluid, bone marrow, ascites, cell lysate. biopsy homogenate, culture supernatant, sewage, and the like. The biological sample may be assayed neat or diluted, or it may be processed to expose nucleic acids, for example, by heating or by incubating the biological sample with a nucleic acid strand- separating reagent or by partial purification. Methods for processing biological samples to expose nucleic acids are known. Preferably, biological samples are partially purified using solid phase extraction prior to subjecting them to the method of the invention. Any solid phase extraction method or combination of methods may be used to perform this partial purification, for example, ion exchange may be used alone or in combination with a desalting method.

In accordance with the invention, an oligonucleotide pharmaceutical and its in vivo degradation products are designated as the "oligonucleotide target" or "target." Any oligonucleotide pharmaceutical may be monitored using the method of the invention, so long as its nucleotide sequence is known or may be determined. Many such oligonucleotide targets are known, for example, deoxyribonucleotides or any combinations of monomers thereof, such monomers being connected together via 5' to 3' linkages which may include any of the linkages that are known in the antisense oligonucleotide art (e.g., phosphorothioates, alkylphosphonates, phosphorodithioates, alkyl phosphonothioates, phosphoramidates, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters). The term oligonucleotide target also encompasses such polymers having chemically modified bases or sugars and/or having additional substituents including without limitation, lipophilic groups, intercalating agents, diamines adamantane and others.

For example, oligonucleotide targets which may be assayed in accordance with the invention may comprise other than phosphodiester internucleotide linkages between the 5' end of one nucleotide and the 3' end of another nucleotide in which the 5' nucleotide phosphate has been replaced with any number of chemical groups, such as a phosphorothioate. Preferably, the phosphorothioate regions of the oligonucleotide targets will have from about 5 to about 24 phosphorothioate-linked nucleosides. The phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054). Oligonucleotide targets with phosphorothioate linkages can be prepared using methods well known in the field such as phosphoramidite

(see, e.g., Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083). or by H- phosphonate (see, e.g., Froehler (1986) Tetrahedron Lett. 27:5575-5578) chemistry. The synthetic methods descπbed in Bergot et al (J Chromatog (1992) 559 35-42) can also be used Examples ot other chemical groups which may occur in oligonucleotide targets include alkylphosphonates, phosphorodithioates, alkyl phosphonothioates, phosphoramidates, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate tπesters or any combinations thereot For example, US Patent No 5.149,797 describes traditional chimeπc oligonucleotide targets having a phosphorothioate core region interposed between methylphosphonate or phosphoramidate flanking regions. U.S. Patent Application Ser No (attorney docket number 47508-559), filed on August 9, 1995 discloses "inverted" chimeric oligonucleotide targets comprising one or more nonionic oligonucleoude region (e.g. alkylphosphonate and or phosphoramidate and/or phosphotnester lntemucleoside linkage) flanked by one or more region of oligonucleotide phosphorothioate Various oligonucleotide targets with modified internucleotide linkages can be prepared according to known methods (see, e.g., Goodchild (1990) Bioconjugate Chem 2:165-187; Agrawal et al., (1988) Proc. Natl. Acad. Sci (USA) 85:7079-7083; Uhlmann et al. (1990) Chem. Rev 90:534-583; and Agrawal et al. (1992) Trends

Biotechnol. 10:152-158

Examples ot modifications to sugars which may be found in oligonucleotide targets include modifications to the position of the πbose moiety which include but are not limited to 2'-O-substιtuted with an -O- lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl, or allyl group having 2-6 carbon atoms wherein such -O-alkyl, aryl or allyl group may be unsubstituted or may be substituted, (e.g., with halo, hydroxy, trifluoromethyl cyano, nitro acyl acyloxy, alkoxy, carboxy, carbalkoxyl, or ammo groups), or with an ammo, or halo group None of these substituuons are intended to exclude the native 2'-hydroxyl group in the case of πbose or 2'-H- in the case ot deoxyπbose PCT Publication No. WO 94/02498 discloses traditional hybnd oligonucleotide targets having regions of 2'-O-subsUtuted πbonucleotides flanking a DNA core region U S. Patent Application Serial No (attorney docket number 47508- 559), filed August 9, 1995, discloses an "inverted" hybrid oligonucleoude target which includes an oligonucleoude compπsing a 2'-O-substιtuted (or 2' OH, unsubstituted) RNA region which is in between two ohgodeoxyπbonucleotide regions, a structure that

"inverted relative to the "traditional" hybnd oligonucleotides.

Other modifications which may occur in oligonucleotide targets include those which are internal or are at the end(s) of the oligonucleotide molecule and include additions to the molecule at the internucleoside phosphate linkages, such as cholesteryl or diamine compounds with varying numbers of carbon residues between the two amino groups, and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the viral genome. Examples of such modified oligonucleotide targets include oligonucleotide targets with a modified base and/or sugar such as arabinose instead of ribose. or a 3', 5'-substituted oligonucleotide having a sugar which, at one or both its 3' and 5' positions is attached to a chemical group other than a hydroxyl or phosphate group (at its 3' or 5' position). Other modified oligonucleotide targets are capped with a nuclease resistance-conferring bulky substituent at their 3' and/or 5' end(s), or have a substitution in one or both nonbridging oxygens per nucleotide. Such modifications can be at some or all of the internucleoside linkages, as well as at either or both ends of the oligonucleotide and/or in the interior of the molecule (reviewed in Agrawal et al. (1992) Trends Biotechnol. 10: 152-158).

Preferably, oligonucleotide targets quantified in accordance with the invention will have from about 12 to about 50 nucleotides, most preferably from about 17 to about 35 nucleotides. Such oligonucleotide targets are preferably complementary to at least a portion of a genomic region, or to a gene or to an RNA transcript thereof such that the oligonucleotide target is capable of hybridizing or otherwise associating with at least a portion of such genomic region, gene or RNA transcript thereof under physiological conditions.

In accordance with the method, the oligonucleotide target comprises a plurality of covalently linked nucleotides, and at least a portion of the internucleotide linkages occur via 5'-to-3' internucleotide linkages as is conventional and as understood in the art. Linear as well as branched oligonucleotide targets are detectable and quantifiable using the method of the invention.

The plurality of covalently linked nucleotides in the target comprises at least four contiguous nucleotides of the oligonucleotide pharmaceutical. Preferably, the plurality of covalently linked nucleotides in the target comprises at least four contiguous nucleotides corresponding to a terminus of the oligonucleotide pharmaceutical.

In one embodiment, suitable for detection of oligonucleotide pharmaceuticals which may have been exposed to 3' exonuclease activity, the plurality of covalently linked nucleotides in the target preferably comprises at least four contiguous nucleotides corresponding to a 5' terminus of the oligonucleotide pharmaceutical. More preferably, the plurality of covalently linked nucleotides in the target comprises at least six contiguous nucleotides corresponding to a 5' terminus of the oligonucleotide pharmaceutical. Most preferably, the plurality of covalently linked nucleotides in the target comprises at least eight contiguous nucleotides corresponding to a 5' terminus of the oligonucleotide pharmaceutical.

As indicated above, oligonucleotide pharmaceuticals may become dephosphorylated in vivo, and therefore in this embodiment of the invention it is necessary to phosphorylate the 5' terminal nucleotides of the oligonucleotide targets present in the biological sample. Methods for phosphorylating oligonucleotides in biological samples are known. For example, oligonucleotide targets may be phosphorylated at their 5' terminal nucleotides using a polynucleotide kinase such as T4 polynucleotide kinase, which is commercially available.

The method of the invention employs two oligonucleotide reagents to monitor or assay the oligonucleotide pharmaceutical. One of the oligonucleotide reagents is designated as an "oligonucleotide bridge." In accordance with the invention, the oligonucleotide bridge comprises a plurality of covalently linked nucleotides, and the linkages of the oligonucleotide bridge are in the 3'-to-5' conformation as is conventional and as understood in the art. The oligonucleotide bridge has a predetermined 5' terminal sequence which is complementary to a plurality of contiguous nucleotides of the oligonucleotide target. The 5' terminal sequence of the oligonucleotide bridge is complementary to at least four contiguous nucleotides of the oligonucleotide target. Preferably, the 5' terminal sequence of the oligonucleotide bridge is complementary to at least four contiguous nucleotides corresponding to a 5' terminus of the oligonucleotide target. More preferably, the 5' terminal sequence of the oligonucleotide bridge is complementary to at least six contiguous nucleotides corresponding to a 5' terminus of the oligonucleotide target. Most preferably, the 5' terminal sequence of the oligonucleotide bridge is complementary to at least eight contiguous nucleotides corresponding to a 5' terminus of the oligonucleotide pharmaceutical. In addition to its predetermined 5' terminal sequence, the oligonucleotide bridge has a predetermined 3' "bridge sequence" comprising a plurality of covalently linked nucleotides.

The second oligonucleotide reagent used in the method of the invention is an "oligonucleotide primer" or "primer" comprising a plurality of covalently linked nucleotides, the linkages of the oligonucleotide primer being in the 5'-to-3' conformation as is conventional and as understood in the art. The primer has a 3' terminal sequence which is complementary to the bridge sequence. In addition to its 3' terminal sequence, the primer comprises a plurality of covalently linked 5' nucleotides forming a 5' terminus. Preferably, the 3' terminus of the primer comprises at least four covalently linked nucleotides. More preferably, the 3' terminus of the primer comprises at least six covalently linked nucleotides. Most preferably, the 3' terminus of the primer comprises at least eight covalently linked nucleotides. The primer is detectable, using any suitable detection means. In some embodiments, the primer is detectable by virtue of at least one label present at its 5' terminus. In these embodiments, any label may be present at the 5' terminus of the primer, at any nucleotide position of the 5' terminus of the oligonucleotide primer. Many suitable labels are available, for example, radioactive labels, fluorescent labels, luminescent labels, magnetic spin labels, enzymatic labels, and the like. When a radioactive label is used it may be necessary to place the label at a non-terminal nucleotide position of the primer, to avoid loss of label through residual enzymatic activity which may be present in the biological sample. The method of the invention may optionally employ a predetermined quantity of a third oligonucleotide, designated the "oligonucleotide internal standard" or "internal standard", to facilitate quantitation of the oligonucleotide pharmaceutical. In accordance with the invention, the internal standard comprises a plurality of covalently linked nucleotides, and the linkages of the internal standard are in the 5'-to-3' conformation as is conventional and understood in the art. The internal standard has a predetermined 5' terminal sequence which is identical to the same contiguous nucleotides of the oligonucleotide target which are complementary to the 5' terminal sequence of the oligonucleotide bridge. Preferably, the 5' terminal sequence of the internal standard is identical to at least four contiguous nucleotides of the oligonucleotide target. More preferably, the 5' terminal sequence of the internal standard is identical to at least six contiguous nucleotides of the oligonucleotide target. Most preferably, the 5' terminal sequence of the internal standard is identical to at least eight contiguous nucleotides of the oligonucleotide target. In accordance with this embodiment of the invention, the internal standard also comprises a means by which it can be physically or chemically differentiated from the oligonucleotide target. Any physical or chemical means may be used to differentiate the internal standard from the oligonucleotide target. For example, the internal standard may contain a larger number of nucleotides, either as a linear molecule or as a branched molecule, than are contained in the oligonucleotide pharmaceutical (the source of the oligonucleotide target). Alternatively, the internal standard may contain chemical groups which cause its overall charge or shape to differ from that of the oligonucleotide target. The means for differentiating the internal standard from the oligonucleotide target may also comprise a label which differs from that which is present on the oligonucleotide primer.

When an internal standard is used in accordance with the invention, it is added to the biological sample, either before or after the phosphorylation step described above. An internal standard added after the phosphorylation step must itself contain a 5' terminal phosphate group.

The structural relationships between the target, oligonucleotide bridge, and primer in this embodiment are schematically depicted in Figure 2, with a representative target having the sequence set forth in SEQ ID NO: l, a representative oligonucleotide bridge having the sequence set forth in SEQ ID NO:2 (noting that SEQ ID NO:2 is set forth in the 5'-to-3' orientation), and a representative primer having the sequence set forth in SEQ

ID NO:3. A representative oligonucleotide internal standard is set forth in SEQ ID NO:4. Of course, the oligonucleotide bridge, primer, and internal standard may have any combination of appropriate sequences, determined by the sequence of the target which is being assayed or monitored. Synthesis of oligonucleotides having any predetermined sequence is well within the level of skill in the art. The oligonucleotide primer and oligonucleotide bridge used in the method of the invention may be made, for example, using a DNA synthesizing machine. Such machines are well known and are commercially available.

In another embodiment, suitable for detection of oligonucleotide pharmaceuticals which may have been exposed to 5' exonuclease activity, the plurality of covalently linked nucleotides in the target preferably comprises at least four contiguous nucleotides corresponding to a 3' terminus of the oligonucleotide pharmaceutical. More preferably, in this embodiment the plurality of covalently linked nucleotides in the target comprises at least six contiguous nucleotides corresponding to a 3' terminus of the oligonucleotide pharmaceutical. Most preferably, in this embodiment the plurality of covalently linked nucleotides in the target comprises at least eight contiguous nucleotides correspondmg to a 3' terminus of the oligonucleotide pharmaceutical.

The structures of the oligonucleotide bridge, oligonucleotide primer, and internal standard used in this embodiment are accordingly altered to correspond with the relevant portions of the oligonucleotide pharmaceutical which would remain after exposure to 5' exonuclease activity. That is, in this embodiment the 3' terminal sequence of the oligonucleotide bridge is preferably complementary to at least four contiguous nucleotides corresponding to a 3' terminus of the oligonucleotide target. More preferably, in this embodiment the 3' terminal sequence of the oligonucleotide bridge is complementary to at least six contiguous nucleotides corresponding to a 3' terminus of the oligonucleotide target. Most preferably, in this embodiment the 3' terminal sequence of the oligonucleotide bridge is complementary to at least eight contiguous nucleotides corresponding to a 3' terminus of the oligonucleotide pharmaceutical. In this embodiment, the bridge sequence is located at the 5' terminus of the oligonucleotide bridge, and the bridge sequence is complementary to a predetermined 5' terminal sequence of the oligonucleotide primer. The label is located at the 3' terminus of the primer, and the 5' terminus of the primer is phosphorylated, in this embodiment. If an internal standard is used in this embodiment, its 3' terminal sequence is identical to the same contiguous nucleotides of the oligonucleotide target which are complementary to the 3' terminal sequence of the oligonucleotide bridge.

In accordance with the method of the invention, an oligonucleotide target, with or without an internal standard, is combined or incubated with an oligonucleotide bridge and an oligonucleotide primer and the three oligonucleotides are incubated together for a time and at a temperature sufficient to allow hybridization to occur. Any reaction conditions may be used to allow hybridization of the target and the primer or of the internal standard and the primer to the oligonucleotide bridge. Nucleic acid hybridization methods are well known, as are methods for optimizing hybridization of nucleic acids having specific sequences, by altering the stringency of the reaction conditions; by adding substances which facilitate nucleic acid hybridization such as formaldehyde, spermine, or spermidine; or by altering the temperature of the hybridization reaction. For example, in accordance with the invention the target, primer, and oligonucleotide bridge or the internal standard, primer and oligonucleotide bridge may be hybridized under stringent (4xSSC at 37°C for ten minutes, 4°C for ten minutes) or relaxed (4xSSC at 50°C or 30-40% formamide at 42°C) conditions.

In accordance with the method of the invention, after hybridization of the target and primer or of the internal standard and primer to the oligonucleotide bridge has occurred, the target, or if present, the internal standard, is ligated to the primer. Any method for ligating contiguous nucleotides may be used in this step of the invention, so long as a covalent bond is formed between the 5' terminal nucleotide of the target and the

3' terminal nucleotide of the primer or between the 5' terminal nucleotide of the internal standard to 3' terminal nucleotide of the primer. Preferably a ligase is used. More preferably, a ligase capable of linking DNA aligned on complementary DNA, RNA aligned on complementary DNA, or DNA aligned on complementary RNA is used. Most preferably, T4 DNA ligase is used. T4 DNA ligase is commercially available, and optimal reaction conditions for ligases are well known.

In accordance with the method of the invention, after ligation of the target or internal standard to the primer has occurred, the ligation product is separated from the oligonucleotide bridge. Separation, in accordance with the invention, includes both denaturation and physical separation of the ligation product from the primer. Any separation method may be used for this step of the method. Preferably, hybridized strands of nucleic acids are denatured using alkaline conditions or by heating, and the denatured strands are physically separated in a flow system such as ultracentrifugation or electrophoresis. More preferably, the ligation product is separated from the oligonucleotide bridge using denaturing capillary gel electrophoresis.

In the last step of the method of the invention, the amount of ligation product, which is indicative of the quantity of the target and therefore of the oligonucleotide pharmaceutical and its degradation products in the biological sample, is measured. Any suitable means for measuring the primer may be used in accordance with the method of the invention. If no label is present on the primer, its quantity may be measured, for example, by determining the ratio of absorbance at 260 nm and 280 nm using ultraviolet absoφtion spectrophotometry, or by comparing the amount of fluorescent dye bound to the ligation product with a standard curve using fluorescence spectroscopy. Alternatively, an unlabeled ligation product may be quantitated using mass spectroscopy. When the primer contains a label, it may be quantitated using a means suitable to the specific label. For example, radioactively labeled ligation products may be quantitated by scintillation counting. Enzymatically labeled ligation products may be quantitated using an assay specific for the enzyme label. Spin labeled ligation products may be quantitated using magnetic resonance methods and apparatus. Luminescently labeled ligation products may be quantitated using suitable luminescence assays and apparatus. Fluorescently labeled ligation products may be quantitated using a suitable means for detecting fluorescent emissions. When an internal standard is used, the concentration of oligonucleotide target is determined relative to amount of ligation product formed from the predetermined quantity of internal standard.

In some embodiments of the invention, the separating and measuring steps of the method are combined. When the oligonucleotide primer is fluorescently labeled, the separating and measuring steps may be combined. A preferred embodiment of the present invention combines capillary gel electrophoresis with laser-induced fluorescence to separate and measure the ligation products in a single step, as is generally described in PCT/US95/01048 and as depicted in Figure 3.

In accordance with the invention, the embodiment suitable for detecting oligonucleotide pharmaceuticals which may have been exposed to 3' exonuclease activity and the embodiment suitable for detecting oligonucleotide pharmaceuticals which may have been exposed to 5' exonuclease activity may be combined to provide a determination of all oligonucleotide species in the biological sample which correspond to the oligonucleotide pharmaceutical. The invention is also embodied in a method of resolving a mixture of homologous oligonucleotides which are different in length. As defined herein, "resolving" means separating the oligonucleotides within the mixture from each other, thereby allowing the different ologonucleotide species present in the mixture to be identified.

As shown in Example 4 and Figure 4. the method of the invention resolved a plurality of fragments of the phosphorothioate oligonucleotide having the sequence set forth in SEQ ID NO: 1. The sample resolved in Example 4 and Figure 4 consisted of a set of nested fragments of an oligonucleotide pharmaceutical, each fragment differing from the next member of the set by a single nucleotide. Figure 4 demonstrates the sensitivity of the method of the invention: homologous 24-mer, 23-mer. 22-mer, 21-mer, 20-mer, 19- mer, 18-mer, 17-mer. 16-mer. 15-mer, 14-mer, 13-mer, 12-mer. 1 1-mer. 10-mer. 9-mer, 8- mer, and 7-mer have been resolved in accordance with this embodiment. Any mixture of homologous oligonucleotides may be resolved in accordance with this embodiment, so long as at least a portion of the homology shared among the oligonucleotide species within the mixture is known or amenable to determination. Preferably, at least four contiguous shared nucleotides of the oligonucleotide species are known or determinable. More preferably, at least six contiguous shared nucleotides of the oligonucleotide species are known or determinable. Most preferably, at least eight contiguous shared nucleotides of the oligonucleotide species are known or determinable. Methods for determining oligonucleotide sequences are well known to those of skill in molecular biology.

The homologous oligonucleotide species within the mixture may contain a phosphate moiety at their 5' termini, or the 5' termini may be unphosphorylated. When the oligonucleotide species do not contain a 5' terminal phosphate moiety, the mixture may be phosphorylated as set forth above, prior to combination with the primer and the oligonucleotide bridge.

When a mixture of homologous oligonucleotides which have been exposed to 3' exonuclease degradation is resolved in accordance with the invention, for example, the 5' phosphate-containing mixture is combined with an oligonucleotide bridge and an oligonucleotide primer, under conditions which allow hybridation to occur. In this embodiment, the oligonucleotide bridge has a predetermined 5' sequence which is complementary to a plurality of the contiguous shared nucleotides of the oligonucleotide species within the mixture. The 5' terminal sequence of the oligonucleotide bridge is complementary to at least four contiguous shared nucleotides of the oligonucleotide species within the mixture. Preferably, the 5' terminal sequence of the oligonucleotide bridge is complementary to at least four contiguous shared nucleotides corresponding to a homologous terminus of the oligonucleotide species within the mixture. More preferably, the 5' terminus of the oligonucleotide bridge is complementary to at least six contiguous shared nucleotides corresponding to a homologous 5' terminus of the oligonucleotide species within the mixture. Most preferably, the 5' terminus of the oligonucleotide bridge is complementary to at least eight contiguous shared nucleotides corresponding to a homologous 5' terminus of the oligonucleotide species within the mixture. As set forth above, the oligonucleotide bridge also has a predetermined 3' bridge sequence, and the oligonucleotide primer has a 3' terminal sequence which is complementary to the bridge sequence. Reagents are altered as set forth above for resolution of mixtures of homologous oligonucleotides which may have been exposed to 5' exonuclease degradation.

In this embodiment, after hybridization between the mixture, the bridge, and the primer occurs and the hybridized oligonucleotide species and primer are ligated. the ligation products are separated from the oligonucleotide bridge and resolved from each other. Preferably separation and resolution of the ligation products occurs in a single step.

The resolved ligation products may optionally be quantitated in this embodiment of the invention. When the primer oligonucleotide is fluorescently labeled, separation, resolution, and quantitation of the ligation products is preferably performed using the combination of capillary gel electrophorosis and laser-induced fluorescence described in PCT US95/01048 and depicted in Figure 3.

The following examples illustrate the preferred modes of making and practicing the present invention, but are not meant to limit the scope of the invention since alternative methods may be used to obtain similar results.

EXAMPLE 1 SAMPLE PREPARATION

Blood was drawn into EDTA-containing vials from a monkey which had received 10 mg kg/day of the oligonucleotide of SEQ ID NO: l for eight weeks. Cells were centrifuged out of the blood sample, and an aliquot of 0.5 ml plasma was diluted 1: 1 with

EQ (0.2 M Tris which had been adjusted to pH 6.3 with H,PO₄, hereinafter referred to as "Tris-PO₄ pH 6.3") and spiked with 10 μl of 5 ppm of the internal standard having the sequence set forth in SEQ ID NO:4, mixed using a Vortex mixer, and centrifuged at room temperature for ten minutes at 14,000 rpm. The sample was filtered through a 2 μm cellulose triacetate filter directly onto an ion exchange cartridge (cat. no. Nucleobond AX-

5, Machery-Nagel GmbH, Germany) which had been pre-equilibrated with two washes of EQ. The cartridge was washed sequentially as indicated below: a. one wash with 0.5 ml EQ; b. three washes with 0.5 ml 0.2 M NaBr, 0.1 M Tris-PO₄ pH 6.3 in 50% formamide (WI); c. one wash with 0.5 ml 0.2 M NaBr, 0.1 M Tris-HCl, pH 7.0 (W2).

The cartridge was then washed once with 0.2 ml 2 M NaBr, 0.1 M Tris-HCl (pH 8.5), prepared in 10% isopropyl alcohol (El). Phosphorothioate oligonucleotides were eluted from the cartridge in 0.5 ml El. The eluant was desalted using ultrafiltration with an 0.5 ml Amicon 3K UF cartridge (Amicon, Inc. Beverly, MA). The ultrafiltration cartridge containing the eluted DNA in El was centrifuged for 30 minutes at 14,000 φm.

EXAMPLE 2 5 -PHOSPHORYLATION OF OLIGONUCLEOTIDES

A 10 μl aliquot of the sample from Example 1 was dialyzed for 60 minutes using drop dialysis (VSWP 0.025 μm filter, Millipore, Bedford, MA). Two μl ligation buffer (cat. no. 202S, New England Biolabs, Beverly, MA) and one μl phosphonucleotide kinase (0.03 u/μl, cat. no. 70031, USB, Cleveland. OH) were added, and the mixture was incubated for 60 minutes at 37 °C.

EXAMPLE 3 HYBRIDIZATION AND LIGATION

To the 13 μl of sample was added two μl ligation buffer (cat. no. 202S, New England Biolabs, Beverly, MA), and one μl of a cocktail containing two ppm primer and four ppm oligonucleotide bridge. The mixture was incubated for ten minutes at 37°C and cooled slowly (over a period of ten minutes) to 4°C. One μl of T4 DNA ligase (1 u/μl, cat. no. 70005, USB, Cleveland, OH) was added and the mixture was incubated for 60 minutes at 37°C. The ligation mixture was dialyzed for 60 minutes using drop dialysis (VSWP 0.025 μm filter, Millipore, Bedford, MA), and analyzed using the laser induced fluorescence/capillary gel electrophoresis apparatus schematically described in Figure 3 and described in detail in Example 4.

EXAMPLE 4

DETECTION OF LIGATION PRODUCT USING LASER INDUCED FLUORESCENCE/GEL ELECTROPHORESIS

A. Preparation of Gel Filled Capillaries Fused-silica capillary tubing (Polymicro Technologies, Phoenix, AZ) with an inner diameter of 75μm, an outer diameter of 375 μm, an effective length of 15-20 cm, and a total length of 30-60 cm is treated with (methylacryloxypropyl) trimethoxysilane (Petrarch Systems. Bristol, PA) and then filled with a degassed solution of polymerizing acrylamide in aqueous or organic solvent (e.g. formamide) media including 0.1-0.3 M Tris-borate, 2- 6 mM EDTA TBE buffer, pH 8.3, containing 6 M to 8.3 M urea). Polymerization was achieved by adding ammonium persulfate solution and TEMED.

B. Laser-Induced Fluorescence/Capillary Gel Electrophoresis Detection

A 30 kV, 500 μA direct current high voltage power supply (Model PS/ER 30P0, 5DM11. Glassman, Whitehouse Station, MD) was used to generate power across the capillary. Laser-induced fluorescence detection employs an argon ion laser (Model 543

100BS, Omnichrom, Chino, CA) with a power supply (Model 155 160, Omnichrom, Chino, CA) mounted on a 4 x 6 foot optical table and operated in the light regulated mode at 0.03 to 0,05 W. The laser light is filtered through a narrow band filter (52640 # 2, Oriel, Stratford, CT), reflected by using a beam steerer (670-TC, 670-BC, Newport, Foundation Valley, CA) and focused into the capillary with a 25 mm focal length lens

(Model KBX043, Newport, Foundation Valley, CA). Sample fluorescence was collected with a 40X microscpe objective (13600, 40x70.65, Oriel, Stratford, CT) and passed through an interference filter (53880 # 16, Oriel, Stratford, CT) and a colored glass filter (LL-500- F, Corion, Holliston MA). A photomultiplier tube (77344, Oriel, Stratford, CT) at 750 V and a photomultiplier readout (Model 7070, Oriel, Stratford, CT) detects the fluorescence.

Data was acquired through an analog-to-digital converter (Model 970, Nelson Analytical, Cupertine, CA) and stored on an AcerPower 486/33 computer (Acer Americal Corp., San Jose, CA).

Figure 4 shows the analysis of the ligation product produced in Example 3 using the apparatus described above and in Figure 3. The electropherogram of Figure 4 shows the ligated 3'-digested degradation products of the oligonucleotide pharmaceutical having the complete sequence set forth in SEQ ID NO: l. Present in the electropherogram are the 25-mer (designated "OP") and the 3'-digested degradation products representing the homologous 5' terminal 24-mer, 23-mer, 22-mer, 21-mer, 20-mer, 19-mer, 18-mer, 17- mer, 16-mer, 15-mer, 14-mer, 13-mer, 12-mer, 11-mer, 10-mer, 9-mer, 8-mer, and 7-mer fragments. The peak corresponding to the internal standard is designated "IS".

Those of skill in the art will recognize that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The presently described embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all variations of the invention which are encompassed within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Cohen, Aharon S. Bourque, Andre J. Wang, Bing

Belenky, Alexei

(ii) TITLE OF INVENTION: METHOD OF MONITORING PHARMACOKINETICS OF OLIGONUCLEOTIDE PHARMACEUTICALS

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Ann-Louise Kemer, Lappin & Kusmer

(B) STREET: 200 State Street

(C) CITY: Boston (D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02109

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICAΗON DATA: (A) APPLICAΗON NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: McDaniels, Patricia A.

(B) REGISTRATION NUMBER: 33,194 (C) REFERENCE/DOCKET NUMBER: HYZ-036

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-330-1300

(B) TELEFAX: 617-330-1311

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "phosphorothioate oligonucleotide"

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: human immunodeficiency virus- 1 gag gene

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CTCTCGCACC CATCTCTCTC CTTCT 25

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide bridge"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TGCGAGAGAC TGCCGG 15

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligonucleotide primer" (xi) SEQUENCE DESCRIPΗON: SEQ ID NO:3:

GTAAAACGAC CGGCAGT 17

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "internal standard"

(iv) ANTI-SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: synthetic oligonucleotide

(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:4: CTCTCGCACC CATCTCTCTT CTTCCCTTCT 30

Claims

What is claimed is:

1. An assay comprising: a) providing a sample containing an unknown quantity of a 5' phosphorylated oligonucleotide target having a known sequence, including a predetermined 5' sequence: b) providing an oligonucleotide bridge having a 5' terminal sequence complementary to the predetermined 5' sequence and a predetermined 3' bridge sequence: c) providing an oligonucleotide primer having a 3' terminal sequence complementary to the bridge sequence; d) combining the sample, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; e) ligating the hybridized target and the primer to create a ligation product; and

0 measuring the amount of ligation product, wherein the amount of ligation product is indicative of the quantity of target in the sample.

2. The assay of claim 1, wherein the primer is labeled.

3. The assay of claim 2, wherein the label is selected from the group consisting of a radioactive label, a fluorescent label, a luminescent label, a magnetic spin label, and an enzymatic label.

4. The assay of claim 3, wherein the label is a fluorescent label.

5. The assay of claim 4, wherein the measuring step f is accomplished by capillary gel electrophoresis and laser-induced fluorescence.

6. The assay of claim 1, wherein the measuring step f comprises the sub-steps of: i. separating the ligation product from the oligonucleotide bridge; and ii. quantifying the ligation product.

7. A method of quantitating a 5'-to-3' linked oligonucleotide target in a biological sample which comprises: a) partially purifying the sample: b) phosphorylating the target at a 5'-terminal nucleotide; c) combining the phosphorylated target with i) an oligonucleotide bridge comprising at least eight 3'-to-5' linked nucleotides, at least four 5'-nucleotides of the bridge having a sequence complementary to at least four 5'-nucleotides of the target; and ii) an oligonucleotide primer having a 3' sequence complementary to a

3'-terminal portion of the bridge; d) incubating the target, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; e) ligating the hybridized target and the primer to create a ligation product; and

8. The method of claim 7, wherein the primer is labeled.

9. The method of claim 8, wherein the label is selected from the group consisting of a radioactive label, a fluorescent label, a luminescent label, a magnetic spin label, and an enzymatic label.

10. The method of claim 9, wherein the label is a fluorescent label.

1 1. The method of claim 10, wherein the measuring step f is accomplished by capillary gel electrophoresis and laser induced fluorescence.

12. The method of claim 7, wherein the measuring step f comprises the sub-sieps of: l. separating the ligation product from the oligonucleotide bridge; and ii. quantifying the ligation product.

13. The method of claim 7, wherein the biological sample is selected from the group consisting of serum, plasma, whole blood, urine, saliva, sputum, milk, lymphatic fluid, lacrimal secretions, cerebrospinal fluid, bone manow, ascites, cell lysate, biopsy homogenate, culture supernatant, and sewage.

14. A method of resolving a mixture of homologous 5' terminal phosphate-containing oligonucleotide species comprising the steps of a) combining the mixture with

(ii) a labeled oligonucleotide primer having a 3' terminal sequence complementary to the bridge sequence; b) incubating the mixture, the bridge, and the primer for a time and at a temperature sufficient to allow hybridization to occur; c) ligating the hybridized species and the primer to create a plurality of ligation products; and d) separating the ligation products from the bridge and from each other.

15. The method of claim 14, further comprising the step of quantitating each ligation product.

16. The method of claim 14, wherein the label is selected from the group consisting of a radioactive label, a fluorescent label, a luminescent label, a magnetic spin label, and an enzymatic label.

17. The method of claim 16, wherein the label is a fluorescent label.

1 . The method of claim 17, wherein the separating step d is accomplished by capillary gel electrophorosis and laser-induced fluorescence.

19. An assay compπsing: a) providing a sample containing an unknown quantity of an oligonucleotide target having a known sequence, including a predetermined 3' sequence; b) providing an oligonucleotide bndge having a 3' terminal sequence complementary to the predetermined 3' sequence and a predetermined 5' bridge sequence; c) providing an oligonucleotide pπmer having a 5' phosphate and a 5' terminal sequence complementary to the bridge sequence; d) combining the sample, the bridge, and the pπmer for a time and at a temperature sufficient to allow hybridization to occur; e) ligating the hybridized target and the primer to create a ligation product; and f) measuring the amount of ligation product, wherein the amount of ligation product is indicative of the quantity of target in the sample.

20. A method of quantitating a 5'-to-3' linked oligonucleotide target in a biological sample which compnses: a) partially purifying the sample: b) combining the target with i) an oligonucleotide bridge comprising at least eight 3'-to-5' linked nucleotides, at least tour 3'-nucleotides of the bndge having a sequence complementary to at least four 3'-nucleotιdes of the target; and ii) an oligonucleotide pnmer having a 5' phosphate and a 5' sequence complementary to a 5'-termιnal portion of the bridge; c) incubating the target, the bridge, and the pnmer tor a time and at a temperature sufficient to allow hybridization to occur; d) ligating the hybridized target and the pnmer to create a ligation product; and e) measuring the amount of ligation product, wherein the amount of ligation product is mdicative of the quantity ot target in the sample.