Compound, method for detecting a nucleic acid target sequence, method for determining if a first nucleic acid and a second nucleic acid hybridize, method for monitoring a nucleic acid amplification reaction and method for detecting amplification of a target sequence

Abstract

 

Description

1 / 99 COMPOUND, METHOD FOR DETECTING A TARGET NUCLEIC ACID SEQUENCE, METHOD FOR DETERMINING WHETHER A FIRST NUCLEIC ACID AND A SECOND NUCLEIC ACID HYBRIDIZE, METHOD FOR MONITORING AN ACID AMPLIFICATION REACTION NUCLEIC AND METHOD FOR DETECTING THE AMPLIFICATION OF A TARGET SEQUENCE CROSS-REFERENCE TO RELATED REQUESTS

[001] This application claims under 35 USC 119(e) the benefit of U.S. Provisional Application No. 61 / 990.913, filed May 9, 2014, which is incorporated herein by reference in its entirety for all purposes. SUMMARY OF THE INVENTION

[002] The present invention provides excited-state energy suppressors, probes and other conjugates comprising these suppressors and methods for their use. Other objects, advantages and aspects of the present invention will be evident from the detailed description below. BRIEF DESCRIPTION OF THE DRAWINGS

[003] Figure 1 shows illustrative compounds of the invention.

[004] Figure 2 is a diagram describing the synthesis of exemplary compounds of the invention.

[005] Figure 3 shows the amplification traces for the Quasar 670-Cosmic Quencher, Quasar 670-BHQ2 and Quasar 670-BBQ probes in a real-time PCR test.

[006] Figure 4 shows the amplification traces for the Quasar 705-Cosmic Quencher and Quasar 705-BHQ2 probes in a real-time PCR test.

[007] Figure 5A - Figure 5B. Figure 5A shows the Petition 870210110461, dated 11 / 29 / 2021, p. 13 / 117 2 / 99 fluorescence intensity for the Quasar 670-Cosmic Quencher, Quasar 670-BHQ2, Quasar 670-BBQ, Quasar 705-Cosmic Quencher, and Quasar 705-BHQ2 probes in a nuclease digestion assay. Figure 5B shows the signal-to-noise ratio for the Quasar 670-Cosmic Quencher, Quasar 670-BHQ2, Quasar 670-BBQ, Quasar 705-Cosmic Quencher, and Quasar 705-BHQ2 probes in a nuclease digestion assay. DETAILED DESCRIPTION OF THE INVENTION Abbreviations

[008] BHQ, as used herein, generally refers to dark suppressants including one or more diazo bonds and, specifically, Black Hole Quenchers™. BHQ examples are described in the Patent of US Patent No. 7,019,129. FET, as used herein, refers to Fluorescence Energy Transfer. FRET, as used herein, refers to Fluorescence Resonance Energy Transfer. These terms are used herein to refer to both radiative and non-radiative energy transfer processes. For example, processes in which a photon is emitted and those involving long-range electron transfer are included under these terms. Throughout this specification, these two phenomena are subsumed under the general term donor-acceptor energy transfer. SNP refers to Single Nucleotide Polymorphism. Definitions

[009] The following definitions are broadly applicable to each of the embodiments of the present invention presented here below. Unless otherwise defined, all technical and scientific terms herein Petition 870210110461, dated 11 / 29 / 2021, p. 14 / 117 3 / 99 used generally have the same meaning as that normally understood by a person skilled in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for the synthesis of nucleic acids and peptides. Molecular biological techniques and procedures are generally performed in accordance with conventional methods in the art and various general references (see generally, Sambrook et al., MOLECULAR CLONING: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, which is incorporated herein by reference). The nomenclature used herein and the laboratory procedures in analytical chemistry and organic synthesis are those well known and commonly employed in the art.Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analysis.

[0010] The term alkyl, by itself or as part of another substituent, means, unless otherwise indicated, a straight-chain or branched-chain hydrocarbon radical, or a combination thereof, which may be fully saturated, mono- or polyunsaturated and may include mono-, di-, tri- and tetravalent radicals having the designated number of carbon atoms (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n Petition 870210110461, dated 11 / 29 / 2021, page 15 / 117 4 / 99 butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologues and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one that has one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethinyl, 1- and 3-propynyl, 3-butynyl and the higher homologues and isomers. The term alkyl, unless otherwise indicated, also optionally includes alkyl derivatives defined in greater detail below, such as heteroalkyl. Alkyl groups that are limited to hydrocarbon groups are called homoalkyl. The term alkyl, as used herein, refers to alkyl, alkenyl, and alkynyl, each of which may be mono-, di-, or polyvalent species, as appropriate to meet valence requirements.The alkyl groups are optionally substituted, for example, with one or more groups referred to here as an alkyl group substituent.

[0011] The term alkylene, by itself or as part of another substituent, means a divalent radical derived from an alkyl radical, as exemplified, but not limited to, by -CH2CH2CH2CH2-, and further includes the groups described below as heteroalkylene. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with groups having 10 or fewer carbon atoms being preferred in the present invention. For alkylene and heteroalkylene linking groups, it is optional that Petition 870210110461, dated 11 / 29 / 2021, p. 16 / 117 5 / 99 no orientation of the linking group is implied by the direction in which the linking group formula is written. For example, the formula -C(O)2R'- represents -C(O)2R'- and optionally -R'C(O)2-. A lower alkyl or lower alkylene is a shorter-chain alkyl group or an alkylene group generally having eight, seven, six, five, or fewer carbon atoms.

[0012] The terms alkoxy, alkylamino and alkylthio (or thioalkoxy) are used in their conventional sense, and refer to alkyl groups attached to the rest of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0013] The term heteroalkyl, by itself or in combination with another term, means, unless otherwise indicated, a stable linear or branched chain, or cyclic alkyl radical consisting of the indicated number of carbon atoms and at least one heteroatom selected from the group consisting of B, O, N, Si and S, wherein the heteroatom may optionally be oxidized and the nitrogen atom may optionally be quaternized. The heteroatom(s) may be placed in any interior position of the heteroalkyl group or at a chain terminal, for example, the position through which the alkyl group is attached to the rest of the molecule. Examples of heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Two or more heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Similarly Petition 870210110461, dated 11 / 29 / 2021, p. 17 / 117 6 / 99 form, the term heteroalkylene alone, or as part of another substituent, refers to a substituted or unsubstituted divalent heteroalkyl radical, as exemplified, but not limited to, by -CH2-CH2-S-CH2-CH2- and CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms may also occupy either or both ends of the chain (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino and the like).

[0014] The terms cycloalkyl and heterocycloalkyl, by themselves or in combination with other terms, represent, unless otherwise indicated, cyclic versions of alkyl and heteroalkyl, respectively. Furthermore, for heterocycloalkyl, a heteroatom may occupy the position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl compounds include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl and the like.

[0015] The terms halo or halogen, by itself or as part of another substituent, means, unless otherwise indicated, an atom of fluorine, chlorine, bromine or iodine. Additionally, terms such as haloalkyl are intended to include mono-haloalkyl and poly-haloalkyl. For example, the term haloalkyl(C1-C4) is intended to include, Petition 870210110461, dated 11 / 29 / 2021, page 18 / 117 7 / 99 but not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl and the like.

[0016] The term aryl means, unless otherwise indicated, a polyunsaturated aromatic substituent, which may be a single ring or multiple rings (preferably 1 to 3 rings, one or more of which is optionally a cycloalkyl or heterocycloalkyl), which are fused together or covalently linked. The term heteroaryl refers to aryl groups (or rings) containing from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) is / are optionally quaternized. A heteroaryl group may be linked to the rest of the molecule via a heteroatom.Examples of non-limiting aryl and heteroaryl groups include phenyl, 1-naphthyl, 2naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. The substituents for each of the above-mentioned aryl and heteroaryl ring systems are selected from the group of aryl group substituents described below.

[0017] For short, the term aryl, when used in combination with other terms (e.g., aryloxy, Petition 870210110461, dated 11 / 29 / 2021, page 19 / 117 8 / 99 arylthioxy, arylalkyl) optionally includes both heteroaryl and homoaryl rings, as defined above. Thus, the term arylalkyl optionally includes those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridylxymethyl, 3-(1-naphthyloxy)propyl and the like).

[0018] Substituents for alkyl and heteroalkyl radicals (including those groups frequently referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) are generically referred to as alkyl group substituents, and they may be one or more of a variety of groups selected from, but not limited to: -OR', =O, =NR', =N-OR', -NR'R, -SR', -halogen, -SiR'RR', OC(O)R', -C(O)R'RR', -CO2R', -CONR'R, -OC(O)NR'R, NRC(O)R', -NR'-C(O)NRR', -NRC(O)2R', -NRC(NR'RR')=NR, -NR-C(NR'R)=NR', -S(O)R', -S(O)2R', S(O)2 NR'R, -NRSO2R', -CN and -NO2, in a number ranging from zero to (2m'+ 1), where m' is the total number of carbon atoms in such a radical.R', R', R'' and R''', in a preferred manner, each independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example, aryl substituted with 1-3 halogens, unsubstituted or substituted alkyl, alkoxy or thioalkoxy, or arylalkyl groups. When a compound of the invention includes more than one group. Petition 870210110461, dated 11 / 29 / 2021, p. 20 / 117 9 / 99 For example, each of the R groups is selected independently, as is each of the R', R', R'', and R'' groups when more than one of these groups is present. When R' and R' are bonded to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, a person skilled in the art will understand that the term alkyl includes groups with carbon atoms bonded to groups other than hydrogen, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, C(O)CF3, -C(O)CH2OCH3, and the like). Exemplary alkyl group substituents include the groups referred to here as reactive functional groups and bonding sites.In several embodiments, the substituent for the alkyl group is a phosphorus-containing group, for example, a phosphodiester or a phosphodiester modification, such as those described herein.

[0019] Similarly to the substituents described for the alkyl radical, substituents for aryl and heteroaryl groups are generically referred to as aryl group substituents. Examples of substituents are selected from the list of alkyl group substituents and others, for example: halogen, -OR', =O, =NR', =N-OR', -NR'R, -SR', -SiR'R', -OC(O)R', -C(O)RR', -CO2R', -CONR'R, -OC(O)NR'R, -NRC(O)R', -NR'-C(O)NRR', -NRC(O)2R', -NR-C (NR'RR')=NR, -NR-C(NR'R)=NR', S(O)R', -S(O)2R', -S(O)2NR'R, -NRSO2R', -CN and -NO2, -R', N3, -CH(Ph)2, fluoroalkoxy (C1-C4), and fluoroalkyl (C1-C4), Petition 870210110461, dated 11 / 29 / 2021, p. 21 / 117 10 / 99 in a number ranging from zero up to the total number of open valences in the aromatic ring system; and where R', R', R'' and R'' are preferably selected independently from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as well as each of the R', R', R'' and R'' groups, when more than one of these groups is present.

[0020] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -TC(O)(CRR')qU-, where T and U are independently -NR-, -O-, CRR'- or a single bond, and eq is an integer from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)rB-, where A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and eq is an integer from 1 to 4. One of the single bonds of the new ring thus formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally replaced with a substituent of the formula -(CRR')sX-(CRR')d-, where sed are independently integers from 0 to 3, and X is O-, -NR', -S-, -S(O)-, -S(O)2- or -S(O)2NR'-.The substituents R, R', R and R' are preferred. Petition 870210110461, dated 11 / 29 / 2021, p. 22 / 117 11 / 99 selected independently of substituted or unsubstituted hydrogen or alkyl (C1-C16). Examples of aryl group substituents include the groups referred to herein as reactive functional groups and binding sites.

[0021] As used herein, the term heteroatom includes oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0022] The symbol R is a general abbreviation representing a substituent group that is selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclyl groups. R may also refer to alkyl group substituents and aryl group substituents.

[0023] The term salt(s) includes salts of compounds that are prepared with relatively non-toxic acids or bases, depending on the particular substituents found in the compounds described herein. When the compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of these compounds with a sufficient quantity of the desired base, either pure or in a suitable inert solvent. Examples of base addition salts include sodium, potassium, calcium, ammonia, organic amino or magnesium salts, or a similar salt. When the compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of these Petition 870210110461, dated 11 / 29 / 2021, page 23 / 117 12 / 99 compounds with a sufficient quantity of the desired acid, either pure or in a suitable inert solvent. Examples of acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydroiodic or phosphorous acids and the like, as well as salts derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, butyric, maleic, malic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic and others like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galacturonic acids and the like (see, for example, Berge et al., Journal of Pharmaceutical Science, 66: 1-19 (1977)).Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into both acid and base addition salts. The hydrates of the salts are also included.

[0024] As used herein, nucleic acid means nucleosides, nucleotides and oligonucleotides, for example, DNA, RNA, whether single-stranded, double-stranded, or more highly aggregated hybridization portions, and any of these chemicals. Modifications include, but are not limited to, those that provide additional chemical groups incorporating charge, polarizability, hydrogen bonding, interaction Petition 870210110461, dated 11 / 29 / 2021, page 24 / 117 13 / 99 electrostatic, and reactivity to nucleic acid ligand nucleobases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, modifications of phosphodiester groups (e.g., phosphorothioates, methylphosphonates), sugar modifications, pyrimidine modifications at position 5, purine modifications at position 8, alterations in exocyclic amines, substitution with non-normalized or non-natural nucleobases such as 4-thiouridine, 5-bromo or 5-iodouracil; skeleton modifications such as peptide nucleic acids (PNA), glycol nucleic acids (GNAs), morpholinos; methylations such as 2'-O-methyl nucleosides, 5-methyl-2'-deoxycytidine; unusual base pairing combinations such as isobases, isocitidine and isoguanidine and the like. A nucleomonomer refers to a single nucleic acid unit that can be a nucleoside, nucleotide, or a modification thereof.

[0025] Nucleobase, as used herein, includes those moieties containing not only the known purine and pyrimidine heterocycles and the pyrimidines of the invention, but also heterocyclic analogs and their tautomers. Purines include adenine and guanine, and exemplary purine analogs include 8-oxo-N6-methyladenine and 7-deazaxanthin. Pyrimidines include thymine, cytosine, and uracil, and their analogs, such as 5-methylcytosine, 5-methyluracil, and 4,4-ethnocytosine. This term also encompasses non-natural nucleobases. Representative non-natural nucleobases include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5 Petition 870210110461, dated 11 / 29 / 2021, page 25 / 117 14 / 99 carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, nitroindole and 2,6-diaminopurine.

[0026] In various embodiments, the compounds of the invention include pyrimidines derivatized at position 5. The derivatives are 1-alkenyl-, 1-alkynyl-, heteroaromatic- and 1-alkynyl-heteroaromatic modifications. 1-Alkenyl means an olefinically unsaturated acyclic group (containing a double bond). 1-Alkynyl means an acetylenically unsaturated acyclic group (containing a triple bond).

[0027] As used herein, nucleoside designates a subset of nucleic acid in which a nucleobase is covalently linked to a sugar or a sugar analog and which optionally includes a phosphite, phosphoramidite, or phosphine. The term nucleoside includes ribonucleosides, deoxyribonucleosides, or any other nucleoside that is an N-glycoside or C-glycoside of a nucleobase. A Petition 870210110461, dated 11 / 29 / 2021, p. 26 / 117 15 / 99 The stereochemistry of the sugar carbons may differ from that of D-ribose. Nucleosides also include those species containing modifications of the sugar portion, for example, where one or more of the hydroxyl groups are substituted with a halogen, a heteroatom, an aliphatic group, or are functionalized as ethers, amines, thiols, and the like. The pentose portion may be substituted with a hexose or an alternative structure, such as a cyclopentane ring, a 6-membered morpholino ring, and the like. Nucleosides, as defined herein, also include a nucleobase linked to an amino acid and / or an amino acid analog having a free carboxyl group and / or a free amino group and / or protected forms thereof. Nucleosides also optionally include one or more nucleobase modifications, for example, modified with a fluorocarbyl, alkenyl, or alkynyl. A nucleoside including a phosphodiester or a phosphodiester modification is referred to herein as a nucleotide.Nucleosides, as defined herein, are also intended to include a nucleobase linked to an amino acid and / or an amino acid analog having a free carboxyl group and / or a free amino group and / or protected forms thereof.

[0028] Sugar modification, as used herein, means any pentose or hexose moiety other than 2'-deoxyribose. Modified sugars include, for example, D-ribose, 2'-O-alkyl, 2'-amino, 2'-halo functionalized pentoses, hexoses, and the like. Examples of sugar modifications include sugars in which one or more of the hydroxyl groups are replaced with a halogen, a heteroatom, an alkyl radical, or are Petition 870210110461, dated 11 / 29 / 2021, p. 27 / 117 16 / 99 functionalized as ethers, esters, and the like. The pentose portion may be replaced by a hexose or an alternative structure, such as a cyclopentane ring, a 6-membered morpholino ring, and the like. Sugars, which have a different stereochemistry from that of D-ribose, are also included.

[0029] Phosphodiester group modification means any analogue of the native phosphodiester group that covalently links adjacent nucleomonomers. Substitutive linkages include phosphodiester analogues, for example, such as phosphorothioate and methylphosphonate, and non-phosphorus-containing linkages, for example, such as acetals and amides.

[0030] Nucleic acid modification also includes 3', 5' and base modifications, such as labeling with an inhibitor (e.g., a BHQ), a fluorophore, an intercalator, a minor groove ligand, a fluorocarbon, a stabilizing group, or another moiety. In several embodiments, the modification, or marker, is covalently conjugated to the oligomer via a linker group.

[0031] Oligomers are defined here as two or more nucleomonomers covalently linked to each other by a phosphodiester or modified phosphodiester moiety. Thus, an oligomer may have as few as two nucleomonomers (a dimer) and essentially no upper limit of nucleomonomers. Oligomers may be linkage-competent and thus may base-pair with cognate single-stranded or double-stranded (or higher-order aggregation) nucleic acid sequences. Oligomers are also useful as synthons for oligomers. Petition 870210110461, dated 11 / 29 / 2021, p. 28 / 117 17 / 99 larger, as described herein. The oligomers may also contain abasic sites and pseudonucleosides. In several embodiments, the oligomers of the present invention are functionalized. The functionalized portions of oligomers are discussed below. When describing certain embodiments, the term oligomer is used interchangeably to refer to the nucleic acid sequence of the oligomer, the modified nucleic acid sequence that provides a probe of the invention, or the modified nucleic acid sequence that provides a solid support of the invention.

[0032] Peptide refers to an oligomer in which the monomers are amino acids and are linked together by amide bonds, alternatively referred to as a polypeptide. When the amino acids are α-amino acids, both the L-optical and D-optical isomers can be used. In addition, non-natural amino acids, for example, β-alanine, phenylglycine, and homoarginine, are also included. Common amino acids that are not encoded by genes can also be used in the present invention. All amino acids used in the present invention can be either the D or L isomer. The L isomers are generally preferred. In addition, other peptidomimetics are also useful in the present invention. For a general review, see Spatola, AF, in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, page 267 (1983).

[0033] A solid support is a solid material possessing a surface for molecular attachment, Petition 870210110461, dated 11 / 29 / 2021, page 29 / 117 18 / 99 compounds, cells or other entities, or of a surface such that the species are attached. The surface of a solid support may be flat or otherwise configured. A solid support may be porous or non-porous. A solid support may be a chip or matrix comprising a surface, and may comprise glass, silicone, nylon, polymers, plastics, ceramics or metals. A solid support may also be a membrane, such as a nylon, nitrocellulose, or polymeric membrane, or a plate or dish, and may be made of glass, ceramics, metals or plastics, such as, for example, a 96-well plate made of, for example, polystyrene, polypropylene, polycarbonate or polyhalomer. A solid support may also be a granule or particle of any shape, and is preferably spherical or nearly spherical, and preferably a granule or particle having a maximum diameter or width of 1 millimeter or less, more preferably between 0.1 and 100 microns.Such particles or granules may consist of any suitable material, for example, glass or ceramic, and / or one or more polymers, such as, for example, nylon, polytetrafluoroethylene, TEFLON™, polystyrene, polyacrylamide, Sepharose, agarose, cellulose, cellulose derivatives or dextran, and / or may comprise metals, particularly paramagnetic metals, such as iron.

[0034] Supports for solid-phase synthesis are known in the art and include, but are not limited to, highly crosslinking polystyrene (McCollum et al., Tetrahedron Lett. 32:4069-4072 (1991), polystyrene / PEG copolymer (Gao, et al., Tetrahedron Lett. 32:5477Petition 870210110461, dated 11 / 29 / 2021, p. 19 / 99 5480 (1991), silica gel (Chow, et al., Nucl Acids Res. 9: 2807-2817 (1981)), silica gel bonded to polyamide (Gait, et al., Nucl Acids Res. 10: 6243-6254 (1982)), cellulose (Crea, et al., Nucl Acids Res. 8: 2331-2348 (1980)) and controlled pore glass (CPG) (Koster, et al., Tetrahedron Lett. 24:747-750 (1983). CPG. CPG beads can be derivatized for the attachment of a nucleomonomer or oligomer in a variety of ways, for example, CPG beads can be treated with 3-aminopropyltriethoxysilane to add an aminopropyl ligand identifier for the attachment of analogous oligonucleotide monomers or dimers (Koster, et al., Tetrahedron Lett. 24:747-750 (1983), or, preferably, a long-chain alkylamine group, more preferably including a terminal nucleoside, can be attached to the CPG (Adams, et al., J. Am. Chem. Soc. 105:661-663 (1983)).

[0035] An intercalator refers to a planar aromatic or heteroaromatic radical that is capable of partial insertion and stacking between adjacent nucleobases. These portions can be small molecules or parts of a larger entity, such as a protein. Examples of non-limiting intercalators include acridines, anthracenes, anthracyclines, anthracyclinone, methylene blue, indole, anthraquinone, quinoline, isoquinoline, dihydroquinones, tetracyclines, psoralens, coumarins, ethidium halides, ethidium homodimers, oxazole yellow. Petition 870210110461, dated 11 / 29 / 2021, p. 31 / 117 20 / 99 homodimeric (YOYO), thiazole orange (TOTO), dynemycins, 1,10-phenanthroline-copper, calqueamicin, porphyrins, distamycins, netropkines and viologens.

[0036] A minor groove ligand refers to a radical typically possessing a molecular weight of about 150 to about 2000 Daltons. The moiety binds in a non-intercalated manner to the minor groove of double-stranded (or higher-order aggregation) DNA, RNA, or their hybrids, preferably with an association constant greater than about 103M-1. Minor groove-binding compounds have widely varying chemical structures; however, exemplary minor groove ligands have an increasing three-dimensional structure. Examples include certain naturally occurring compounds such as netopsin, distamycin and lexitropsin, mithramycin, cromomycin A3, olivomycin, anthramycin, sibiromycin, as well as related synthetic antibiotics and derivatives.Certain heterocyclic biquaternary ammonium compounds, diarylamidines such as pentamidine, stilbamidine and berenil, CC-1065 and pyrroloindole and related indole polypeptides, Hoechst 33258, 4'-6-diamidino-2-phenylindole (DAPI), as well as a series of oligopeptides consisting of naturally occurring or synthetic amino acids, are minor groove ligand compounds. Exemplary minor groove ligands are described in U.S. Patent No. 6,084,102. This type of linkage can be detected by well-established spectrophotometric methods such as ultraviolet (UV) light spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, and also by gel electrophoresis. Shifts in UV spectra. Petition 870210110461, dated 11 / 29 / 2021, page 32 / 117 21 / 99 by means of binding of a minor groove ligand molecule and NMR spectroscopy using the Nuclear Overhauser effect (NOSEY) are particularly well-known and useful techniques for this purpose. Gel electrophoresis detects the binding of a minor groove ligand to double-stranded DNA or its strand fragment, because, by means of this binding, the mobility of the double-stranded DNA is altered.

[0037] The minor groove ligand is typically attached to the oligomer or solid support via a ligand comprising a chain of about 20, about 15, about 10, or about 5 atoms.

[0038] Intercalating moieties or agents are easily distinguished from minor groove ligands based on the fact that intercalating agents are planar aromatic molecules (preferably polycyclic) and the minor groove ligands have an increasing shape or analogous geometry. An experimental distinction can also be made by NMR spectroscopy using the Nuclear Overhauser effect.

[0039] The term ligand or L, as used herein, refers to a single covalent bond (zero order) or a series of stable covalent bonds incorporating 1-30 non-hydrogen atoms selected from the group consisting of C, N, O, S, Si and P, which covalently link together the components of the compounds of the invention, for example, linking a solid support to a stabilizing agent, an inhibitor, an oligomer or nucleomonomer of the invention; or linking a suppressor or stabilizing portion of a nucleobase of Petition 870210110461, dated 11 / 29 / 2021, p. 33 / 117 22 / 99 an amidite of the invention. Exemplary ligands include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 atoms other than hydrogen. Unless otherwise indicated, ligand, ligand, bond, conjugation, conjugate and analogous terms relating to attachment refer to techniques using and species incorporating ligands. Exemplary ligands include a binding site as defined herein. In addition, a ligand is used to attach an oligomer or nascent oligomer (during oligomer synthesis) to the solid support of the invention. Thus, the invention also provides an oligomer of the present invention covalently linked to a solid support (for example, a solid support of the invention) through a binder.The solid and oligomeric supports of the present invention optionally include a cleavable linker between two components of the solid support and the oligomer (for example, between the oligomer and the solid support, between the fluorophore and the oligomer, between the suppressor and the oligomer, between the fluorophore and the suppressor, etc.). In several embodiments, the linker that joins the solid support to the oligomer is a cleavable linker.

[0040] A cleavable linker is a linker that has one or more cleavable groups that can be broken as a result of a reaction or condition. An exemplary cleavable linker is located at R8 of Formula I or II, which serves to allow the convenient separation of a synthesized oligomer of the invention from the solid support on which it was synthesized. The term cleavable group refers to a unit that allows the release of a component from Petition 870210110461, dated 11 / 29 / 2021, page 34 / 117 23 / 99 solid support or oligomer of the present invention by cleaving a bond that joins the released portion to the rest of the conjugate. Examples of cleavage mechanisms used in both the preparation and use of the oligomers and solid supports of the invention are enzymatically mediated or otherwise chemically mediated.

[0041] In addition to enzymatically cleavable groups, it is within the scope of the present invention to include one or more sites that are cleaved by the action of an agent other than an enzyme. Exemplary non-enzymatic cleavage agents include, but are not limited to, acids, bases, light (e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxycinnamate esters, benzoin esters) and heat. Many cleavable groups are known in the art. See, for example, Jung et al., Biochem. Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem., 265:14518-14525 (1990); Zarling et al., J. Immunol., 124:913-920 (1980); Bouizar et al., Eur. J. Biochem, 155:141-147 (1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J. Immunol., 143:1859-1867 (1989). Furthermore, a wide range of cleavable, bifunctional spacer arms (both homo- and hetero-bifunctional) are commercially available.

[0042] An exemplary cleavable group is cleavable by a reagent, for example, sodium hydroxide, ammonia, or another amine. In several embodiments, the cleavable ligand is readily cleaved at room temperature or upon heating. In one embodiment, R8 of Formula I or II comprises a cleavable ligand that is cleaved by treatment with an amine, for example, ammonia or another amine. Petition 870210110461, dated 11 / 29 / 2021, page 35 / 117 24 / 99 essentially anhydrous, in an organic solvent.

[0043] A binding site is a portion that links two or more components (e.g., functional component, solid support, oligonucleotides, or ligands). This term refers to a covalent bond that is formed by the reaction of complementary reaction partners, each of which has a functional group with complementary reactivity to that of its partner. Binding sites on the solid support and oligomers of the present invention are selected independently. Exemplary linkage sites include, but are not limited to, S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH and NHC(O)O and OC(O)NH, CH2S, CH2O, CH2CH2O, CH2CH2S, (CH2)oO, (CH2)oS or (CH2)oYx-PEG where Yx is S, NH, NHC(O), C(O)NH, NHC(O)O, OC(O)NH or O and o is an integer from 1 to 50. In each of these exemplary linkage sites, NH may be NRt., where Rt is substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. A linkage site may also be a phosphodiester group.In various embodiments, the binding site is located between a ligand and a fluorophore, a ligand and a suppressor, a ligand and a stabilizing moiety, or a linking element and a solid support. In an illustrative embodiment of the oligomers and solid support of the invention, each binding site is different.

[0044] The term fluorophore, as used herein, refers to a portion that is inherently fluorescent or exhibits a change in fluorescence upon binding to a biological compound or metal ion, or by a metabolic enzyme, i.e., fluorogenic. Fluorophores can be substituted to alter solubility, properties Petition 870210110461, dated 11 / 29 / 2021, p. 36 / 117 25 / 99 spectral and physical properties of the fluorophore. Numerous fluorophores are known to those skilled in the art and include, but are not limited to, coumarins, acridines, furans, dansyls, cyanines, pyrenes, naphthalenes, benzofurans, quinolines, quinazolinones, indoles, benzazoles, borapoliazaindacenes, oxazines and xanthines, the latter including fluoresceins, rhodamines, rosamines and rhodoils. These and other fluorophores used in the present invention are described in Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS. Furthermore, useful fluorophores are described in the U.S. Common Property Patent Application Publications Nos. 2005 / 0214833 and 2005 / 0170363 and hereafter.

[0045] As used herein, suppressor refers to any fluorescence-modifying unit of the invention that can attenuate, at least partially, the light emitted by a fluorophore. This attenuation is referred to herein as suppression. Thus, in various embodiments, excitation of the fluorophore in the presence of the suppression group leads to an emission signal that is less intense than expected or even completely absent. Suppression typically occurs through energy transfer between the excited fluorophore and the suppression group.

[0046] The fluorophore or suppressor may include substituents that enhance a desirable property, for example, water solubility, cell permeability, or an altered absorption and emission spectrum, relative to the parent compound in the absence of such a substituent. As such, the fluorophore or suppressor used in Petition 870210110461, dated 11 / 29 / 2021, page 37 / 117 26 / 99 inventions include substituents that improve a desirable property relative to an identically derived compound, in the absence of the improving substituent.

[0047] A functional component is a generic term for a portion of a compound of the present invention having a structure selected from a suppressor, a fluorophore, or a stabilizing moiety (including, but not limited to, intercalators, minor groove linking moieties, nucleobases modified with a stabilizing moiety (e.g., alkynyl moieties and fluoroalkyl moieties), and conformational stabilizing moieties, as described in the U.S. Patent Application Publication No. 2007 / 0059752).

[0048] The term “polynucleotide amplification” includes, but is not limited to, methods such as polymerase chain reaction (PCR), ligase amplification (or ligase chain reaction, LCR), and Q-beta replicase-based amplification methods. These methods are well known and widely practiced in the art. See, for example, U.S. Patents Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al., 1989a (for LCR). Reagents and hardware for performing PCR are commercially available. (m) be cloned(s) before analysis Petition 870210110461, dated 11 / 29 / 2021, page 38 / 117 27 / 99 sequence. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments was described by Scharf (1986). The present invention provides oligomeric primers for use in amplification processes. In addition, a solid support for use in the synthesis of such primers is provided. Besides primers, the invention provides probes and methods for using such probes to detect, characterize and / or quantify the amplification products; solid supports for use in synthesizing these oligomeric probes are also provided.

[0049] The term base stacking disturbances refers to any event that causes a disturbance in base stacking, such as, for example, a base pair mismatch, a protein binding to its recognition site, or any other entities that form oligonucleotide adducts. Several probes of the invention are capable of detecting, characterizing, and / or quantifying such base stacking disturbances. Furthermore, the invention provides the use of solid supports in the synthesis of probes capable of detecting, characterizing, and / or quantifying such base stacking disturbances.

[0050] The term hybridization refers to two nucleic acid chains associated with each other, which may or may not be fully base-paired: generally, this term refers to an association including an oligomer of the present invention, whether attached to a solid support or in solution.

[0051] The term denaturant refers to the process by which Petition 870210110461, dated 11 / 29 / 2021, p. 39 / 117 28 / 99 which nucleic acid duplex chains (or higher-order aggregates) are no longer base-paired by hydrogen bonding and are separated into single-stranded molecules. Denaturation methods are well known to those skilled in the art and include thermal denaturation and alkaline denaturation. This term generally refers to the dissociation of a probe of the invention from its target nucleic acid.

[0052] The term mismatches refers to nucleic acid nucleobases hybridized in nucleic acid duplexes (or higher-order aggregates) that are not 100% complementary. Mismatches include any incorrect pairing between the nucleobases of two nucleobases located on complementary nucleic acid strands that are not Watson-Crick base pairs, for example, A:T or G:C. The lack of full homology may be due to deletions, insertions, inversions, substitutions, or frameshift mutations. In several embodiments, the oligomer of the invention includes a mismatch with respect to its target nucleic acid, preferably allowing detection and / or characterization and / or quantification of the corresponding mismatch in its target. In certain embodiments, the mismatch is a single nucleotide mismatch.

[0053] As used herein, the term polymorphism refers to a variation in the sequence of a gene, and mutation refers to a variation in the sequence of a gene that is associated, or believed to be associated, with a phenotype. The term gene refers to a segment of Petition 870210110461, dated 11 / 29 / 2021, p. 40 / 117 29 / 99 coding of the genome for a control region of the functional product protein. The polymorphic markers used according to the present invention for individual identification can be located in coding or non-coding regions of the genome, and various probes of the invention are designed to hybridize with nucleic acid regions, including these markers. The term individual, as used herein, refers to an individual providing a test sample from which target nucleic acids are obtained for the purpose of genetic testing. The oligomers of the present invention are for use in the detection and / or characterization and / or quantification of polymorphisms and mutations. In addition, the solid supports of the invention are for use in the synthesis of oligomers for use in the detection and / or characterization and / or quantification of polymorphisms and mutations.

[0054] The term probe, as used herein, refers to nucleic acid oligomers prepared using a solid support or amidite of the invention. In various embodiments, the probes produce a detectable response after interaction with a binding partner. The probes include at least one detectable portion, or a pair of portions that form a detectable energy transfer pair at some state change of the probe in response to its interaction with a binding partner. The present invention provides probes and amidites and solid supports for use in synthesizing probes. Exemplary probes of the invention are for use in detecting a polymorphism. In various embodiments, the polymorphism is a single nucleotide polymorphism (SNP). Petition 870210110461, dated 11 / 29 / 2021, p. 41 / 117 30 / 99

[0055] The term detectable response, as used herein, refers to a change, or occurrence, in a signal that is directly or indirectly detectable, either through observation or by instrumentation, and the presence of, or preferably the magnitude of, a target binding partner for a probe in the test sample. Typically, the detectable response is an optical response from a fluorophore that results in a change in the wavelength distribution patterns or absorbance or fluorescence intensity, or a change in light scattering, fluorescence quantum yield, fluorescence lifetime, fluorescence polarization, a change in excitation or emission wavelength, or a combination of the above parameters. The detectable change in a given spectral property is generally an increase or a decrease in fluorescence intensity.However, spectral changes that result in a shift in the emission or excitation wavelength of fluorescence are also useful. The change in ion-binding fluorescence is generally due to conformational or electronic changes in the indicator that can occur in either the excited or ground state of the fluorophore, due to changes in electron density at the ion-binding site, due to quenching of fluorescence by the bound target metal ion, or due to any combination of these or other effects. Alternatively, the detectable response is an occurrence of a signal where the fluorophore is inherently fluorescent and does not produce a change in signal after the ion binding. Petition 870210110461, dated 11 / 29 / 2021, page 42 / 117 31 / 99 binding to a metal ion or a biological compound. The present invention provides probes that provide a detectable response and solid supports for use in synthesizing such probes.

[0056] 0 The term carrier molecule, as used herein, refers to any molecule to which a compound of the invention is attached. Representative carrier molecules include a protein (e.g., enzyme, antibody), glycoprotein, peptide, saccharide (e.g., mono-, oligo- and polysaccharides), hormone, receptor, antigen, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc., without limitation. Carrier molecule also refers to species that cannot be considered as falling within the classical definition of a molecule, for example, solid support (e.g., synthesis support, chromatographic support, membrane), viruses and microorganisms.

[0057] The jwv symbol, shown perpendicular to a bond, indicates the point where the shown portion is attached to the rest of the molecule.

[0058] In some forms, the definition of the terms used here is in accordance with IUPAC. Cosmic Suppressors

[0059] In one aspect, the invention provides a compound (suppressant) having a structure according to Formula I or II: Petition 870210110461, dated 11 / 29 / 2021, p. 43 / 117 32 / 99

[0060] Rla, Rlb, R7ae R7b are selected independently from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted heterocycloalkyl; Rlae Rlb, together with the carbon atom to which they are attached, are optionally joined to form a ring that is a member selected from substituted or unsubstituted C3-C7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl; R7ae R7b, together with the carbon atom to which they are attached, are optionally joined to form a ring that is a member selected from substituted or unsubstituted C3-C7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl; At least one of Rla, Rlb, R7ae, and R7b is not H; Rsi „ I— L*s R8 is selected from H, * and R*. Lx, Lxs, Rs and Rxs are as defined here. [ 0 0 61 ] Any of the combinations of R1a, R1b, R7a, R7b, and R8 are encompassed by this disclosure and specifically provided by the invention. Petition 870210110461, dated 11 / 29 / 2021, p. 44 / 117 33 / 99

[0062] In some embodiments, R1 and R1b are each H. In some embodiments, R1 and R1b are independently selected from unsubstituted C1, C2, C3, C4, C5 and C6 alkyl groups. In some embodiments, R1 and R1b are each methyl.

[0063] In some embodiments, R7ae R7bsion, each, H. In some embodiments, R7ae R7bsion are independently selected from unsubstituted C1, C2, C3, C4, C5 and C6 alkyl groups. In some embodiments, R7ae R7bsion, each, methyl. [00e4]In some embodiments, R1 and R1b are independently selected from unsubstituted C1, C2, C3, C4, C5 and C1 alkyl groups, and R7 and R7b are each H-groups. In some embodiments, R1 and R1b are each methyl, and R7 and R7b are each H-groups. In some embodiments, R1 and R1b are each H-groups, and R7 and R7b are independently selected from unsubstituted C1, C2, C3, C4, C5 and C1 alkyl groups. In some embodiments, R1 and R1b are each H-groups, and R7 and R7b are each methyl. In some embodiments, R1a, R1b, R7 and R7b are independently selected from unsubstituted C1, C2, C3, C4, C5 and C1 alkyl groups. In some forms, R1a, R1b, R7a and R7b are each methyl. [00e5]In some embodiments, Lx is selected from a substituted or unsubstituted alkyl linkage, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl. In some embodiments, Lx is selected from unsubstituted C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10 alkyl. [00ee]In some modalities, Lxsé selected a Petition 870210110461, dated 11 / 29 / 2021, p. 45 / 117 34 / 99 from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl and substituted or unsubstituted heterocycloalkyl. In some embodiments, Lxsé substituted heteroalkyl.

[0067] In some embodiments, Rs is selected from a protected or unprotected functional reactive group, a binding site, and a solid support. In some embodiments, Rs is selected from -OH, -ODMT, and a binding site covalently linked to a ligand on a solid support. DMT refers to 4,4'-dimethoxytrityl. In some embodiments, the solid support is controlled pore glass (CPG).

[0068] In some embodiments, Rx is selected from a protected or unprotected reactive functional group and a binding site. In some embodiments, Rx is selected from a phosphoramidite, -OH, -ODMT, COOH, an active ester (such as an N-hydroxysuccinimide (NHS) ester) and -NH2. DMT refers to 4,4'-dimethoxytrityl.

[0069] In some embodiments, the binding site is covalently linked to an independently selected member of a nucleoside, a nucleoside ligand, a nucleotide, a nucleotide ligand, an oligonucleotide, an oligonucleotide ligand, a nucleic acid, a nucleic acid ligand, a carrier molecule, a carrier molecule ligand, a solid support, and a solid support ligand.

[0070] In some modalities, Rx is a linking site Petition 870210110461, dated 11 / 29 / 2021, p. 46 / 117 35 / 99 covalently linked to a ligand to a given nucleoside having the structure: where the ring marked B is a nucleobase; Rns θ -OH or a phosphoramidite; and Rn5e -OH or -ODMT. DMT refers to 4,4'-dimethoxytrityl.

[0071] In some modes, R8 is selected from: O ODM' q .eNC(CH2hOF''NPr2 Monomers

[0072] In various embodiments, the invention provides monomeric nucleic acids for use in the synthesis of oligomers with an internal modification. In a representative embodiment, the monomeric nucleic acid carries a suppressor moiety. A monomeric nucleic acid Petition 870210110461, dated 11 / 29 / 2021, p. 47 / 117 36 / 99 is an example according to this modality, it has the formula: where Q is a suppressor portion; Lné is a ligand; The ring marked B is a nucleobase; Rn3 θ -OH or a phosphoramidite; and Rn5e -OH or -ODMT. DMT refers to 4,4'-dimethoxytrityl.

[0073] In some forms, the suppressor portion (Q) has a structure according to Formula I or II: wherein, R1a, R1b, R7a and R7b are as defined herein; R8 comprises a binding site covalently linked to Ln.

[0074] In some embodiments, Lné is selected from a linkage, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, heteroalkyl Petition 870210110461, dated 11 / 29 / 2021, p. 48 / 117 37 / 99 substituted or not substituted and substituted or not substituted heterocycloalkyl.

[0075] Examples of nucleobases include: Oligomers

[0076] A nucleic acid oligomer, for example, a probe, prepared using a monomer of the invention and including the components of the monomer from which it is synthesized, is also provided. [ 0077]Exemplary oligomers include oligonucleotides, oligonucleosides, oligodeoxyribonucleotides containing (2'-deoxy-D-ribose or modified forms thereof), i.e., DNA, oligoribonucleotides (containing D-ribose or modified forms thereof), i.e., RNA, and any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine nucleobase, or modified purine or pyrimidine nucleobases. Oligomer, as used herein, also includes compounds in which adjacent nucleomonomers are linked via amide bonds, as previously described (Nielsen et al., Science (1991) 254:1497-1500). The elements normally found in oligomers, such as the furanose ring and / or the phosphodiester linkage, can be replaced by any suitable functionally equivalent element.Oligomer is therefore intended to include any structure that serves as a support or backing for the nucleobases in which the scaffolds form. Petition 870210110461, dated 11 / 29 / 2021, page 49 / 117 38 / 99 allow binding to target nucleic acids in a sequence-dependent manner.

[0078] Examples of groups linking nucleomonomers in an oligomer of the present invention include (i) phosphodiester and phosphodiester modifications (phosphorothioate, methylphosphonate, etc.), (ii) substitution linkages containing a non-phosphorous isostere (formacetal, riboacetal, carbamate, etc.), (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose instead of ribose or deoxyribose, and (iv) nucleomonomers linked via amide linkages or acyclic nucleomonomers linked via any suitable substituent linkage.

[0079] The oligomers of the present invention can be formed using modified and conventional nucleomonomers and synthesized using solid-phase (or solution-phase) and standard oligomer synthesis techniques, which are now commercially available. In general, the oligomers can be synthesized by a method comprising the steps of: synthesizing a nucleomonomer or oligomer synthon having a protecting group and a nucleobase and a coupling group capable of coupling to a nucleomonomer or oligomer; coupling the nucleomonomer or oligomer synthon to an acceptor nucleomonomer or an acceptor oligomer; removing the protecting group; and repeating the cycle as necessary until the desired oligomer is synthesized.

[0080] The oligomers of the present invention can be of any length, including those larger than 40, 50 or 100 nucleomonomers. In various embodiments, the Petition 870210110461, dated 11 / 29 / 2021, page 50 / 117 39 / 99 oligomers contain 2-100 nucleomonomers. Lengths greater than or equal to about 10 to 40 nucleomonomers are useful for therapeutic or diagnostic applications. Short oligomers containing 2, 3, 4, or 5 nucleomonomers are specifically included in the present invention and are useful, for example, as synthons.

[0081] Oligomers having a random sequence and containing fewer than 20, fewer than 15, or fewer than 10 nucleomonomers are useful for primers, for example, in cloning or amplification protocols that use random sequence primers, provided that the oligomer contains residues that can serve as a primer for polymerases or reverse transcriptases.

[0082] Oligomers may contain conventional phosphodiester linkages or may contain phosphodiester modifications, such as phosphoramidate linkages. These substitution linkages include, but are not limited to, embodiments in which a portion of the formula -OP(O)(S)-O(phosphorothioate), -OP(S)(S)-O- (phosphorodithioate), -OP(O)-(NRo2)-X-, -OP(O)(Ro)-OOP(S)(Ro)-O- (thionoalkylphosphonate), -P(O)(ORp)-X-, -OC(O)-X- or -OC(O)(NRp2)-X-, wherein Ro is H (or a salt) or alkyl (C1-12) and R is an alkyl group (C1-9) and the linkage is joined to adjacent nucleomonomers through an -O- or -S- bonded to a carbon of the nucleomonomer. In various embodiments, the surrogate linkages for use in the oligomers of the present invention include phosphodiester, phosphorothioate, methylphosphonate, and thionomethylphosphonate linkages. Phosphorothioate and methylphosphonate linkages confer additional stability to the oligomer in physiological environments. Although Petition 870210110461, dated 11 / 29 / 2021, p. 51 / 117 40 / 99 Although not all of these linkages in the same oligomer need be identical, particularly preferred oligomers of the invention contain uniformly phosphorothioate linkages or uniformly methylphosphonate linkages.

[0083] Oligomers, or segments thereof, are conventionally synthesized and can be prepared using a compound of the invention. The synthetic methods known in the art and described herein can be used to synthesize oligomers containing the compounds of the invention, as well as other nucleobases known in the art, using appropriately protected nucleomonomers. Methods for the synthesis of oligomers are found, for example, in Froehler, B., et al., Nucleic Acids Res. (1986) 14:5399-5467; Nucleic Acids Res. (1988) 16:4831-4839; Nucleosides and Nucleotides (1987) 6:287-291; Froehler, B., Tetrahedron Lett. (1986) 27:5575-5578; Caruthers, MH on Oligodeoxynucleotides-Antisense Inhibitions of Gene Expression (1989), JS Cohen, editor, CRC Press, Boca Raton, pages 7-24; Reese, CB et al., Tetrahedron Lett. (1985) 26:2245-2248. The synthesis of methylphosphonate-linked oligomers via methylphosphoramidite chemistry has also been described (Agrawal, S. et al., Tetrahedron Lett. (1987) 28:3539-3542; Klem, RE, et al., International Publication Number WO 92 / 07864).

[0084] As described herein, the invention provides oligomer conjugates. For example, the oligomers can be covalently linked to various functional components, such as stabilizing moieties, fluorophores, deactivators, intercalators, and substances that specifically interact with the minor groove of DNA. Petition 870210110461, dated 11 / 29 / 2021, p. 52 / 117 41 / 99 double helix (minor groove ligands, MGB). Other selected portions of the conjugate may be radioactive markers, such as fluorescent portions, enzymes, or groups that facilitate cell association using cleavage ligands and the like. Suitable radioactive markers include 32P, 35S, 3H, and 14C; and suitable fluorescent markers include fluorescein, resorufin, rhodamine, BODIPY (Molecular Probes), and Texas red; suitable enzymes include alkaline phosphatase and horseradish peroxidase. Additional fluorophores are presented here and are generally recognized in the art. Other covalently linked portions include biotin, antibodies or antibody fragments, and proteins, for example, transferrin and the HIV Tat protein.

[0085] As discussed herein and recognized in the art, oligomers can be derivatized through any convenient linkage. For example, minor groove ligands, fluorophores, deactivators, and intercalating agents, such as acridine or psoralen, can be linked to the oligomers of the present invention by means of any available -OH or -SH, for example, at the 5' terminal position of the oligomer, at the 2' positions of RNA, or an OH, NH2, COOH, or SH incorporated at the 5' position of pyrimidines. A derivatized form containing, for example, -CH2CH2NH2, CH2CH2CH2OH, or -CH2CH2CH2SH at the 5' position is of use in the present invention. Conjugates including polylysine or lysine can be synthesized, as described, and can further improve the binding affinity of an oligomer to its target nucleic acid sequence (Lemaitre, M. et al., Proc Natl Acad Sci USA (1987) 84: 648-652; Lemaitre, Petition 870210110461, dated 11 / 29 / 2021, page 53 / 117 42 / 99 M. et al., Nucleosides and Nucleotides (1987) 6:311-315).

[0086] A wide variety of substituents can be attached, including those linked via substituent links or linkages. The -OH radicals in the phosphodiester bonds of oligomers can be replaced by phosphate groups, protected by conventional protecting groups, or coupling groups to prepare additional linkages to other nucleomonomers, or they can be linked to the conjugated substituent. The 5' OH terminal can be phosphorylated; the 2'-OH or OH substituents at the 3' terminal can also be phosphorylated. Hydroxyl groups can also be derivatized to standard protecting groups.

[0087] The oligomers of the invention can be covalently derivatized into portions that facilitate cell association using cleavable ligands. The ligands used for such conjugates may include disulfide bonds, which are reduced after the oligomer carrier conjugate has entered a cell. Disulfide-containing ligands of this type have a controllable half-life. These ligands are stable under extracellular conditions relative to intracellular conditions due to the redox potential of the disulfide bond. Donor and Recipient Portions Suppressors

[0088] Illustrative solid and oligomeric supports of the present invention include an inhibitor covalently linked thereto, optionally via a ligand. In various embodiments, the suppressor is a moiety having a structure according to Formula I or II: Petition 870210110461, dated 11 / 29 / 2021, page 54 / 117 43 / 99 wherein R1a, R1b, R7a, R7b and R8 are as defined herein; and R8 comprises a binding site covalently linked (directly or through a ligand) to a solid support, a binding site covalently linked (directly or through a ligand) to an oligomer, or both.

[0089] One of the advantages of the compounds of the invention is that a wide range of energy-donating molecules can be used in conjunction with the solid supports and functionalized oligomers as suppressors. A vast variety of fluorophores is known to those skilled in the art. See, for example, Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); Dexter, DL, J. of Chemical Physics 21:836-850 (1953); Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992); Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I. Ann. Rev. Biochem., 40:83-114 (1971); Stryer, L. Ann. Rev. Biochem., 47:819-846 (1978); Wang et al., Tetrahedron Letters 31:6493-6496 (1990); Wang et al.. Anal. Chern. 67:1197-1203 (1995).

[0090] A non-exhaustive list of examples of donors Petition 870210110461, dated 11 / 29 / 2021, pp. 55 / 117 44 / 99 which can be used in conjunction with the suppressors of the invention is provided in Table 1.

[0091] TABLE 1 Suitable portions that can be selected as donors or acceptors in donor-acceptor energy transfer pairs. 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid Acridine and its derivatives: acridine acridine isothiocyanate 5-(2'-Aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS) 4-Amino-N-[3-vinylsulfonyl)phenyl]naphthalimide 3,5-Disulfonate N-(4-anilino-1-naphthyl)maleimide Anthranilamide BODIPY Bright yellow Coumarin and derivatives: Coumarin 7-amino-4-methylcoumarin (AMC, Coumarin 120) - amino-4-trifluoromethylcouluarin (Cumaran 151) Cyanine dyes Cyanine 4',6-diaminidino-2-phenylindole (DAPI) 5',5'-dibromoprogalol-sulfonaphthalein (Red of Bromopyrogallol) 7-Diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin Diethylenetriamine pentaacetate 4,4'-diisothiocyanatodihydro-stilbene-2,2'-Acid Petition 870210110461, dated 11 / 29 / 2021, page 56 / 117 45 / 99 disulfonic acid 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid 5-[dimethylamino]-naphthalen-1-sulfonyl chloride (DNS, dansyl chloride) 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) 4-Dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC) Eosin and derivatives: eosin isothiocyanate Erythrosine and its derivatives: erythrosine B erythrosine isothiocyanate Etídio Fluorescein and derivatives: 5-carboxyfluorescein (FAM) 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF) 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) fluorescein fluorescein isothiocyanate QFITC ​​(XRITC) Fluorescamine IR144 IR1446 Malachite Green 4-Methylumbelliferone Isothiocyanate Ortho-cresolphthalein Nitrotyrosine Paraosaniline Phenol Red Petition 870210110461, dated 11 / 29 / 2021, pp. 57 / 117 46 / 99 B-phycoerythrin o-Phthaldialdehydes Pyrene and derivatives: pyrene butyrate pyrene 1-pyrene butyrate succinimidyl Quantum dots Reactive Red 4 (Bright Red 3B-A Cibacron™) Rhodamine and its derivatives: 6-carboxy-X-rhodamine (ROX) 6-carboxyrodamine (R6G) lissamine chloride rhodamine rhodamine B sulfonyl (Rhod) rhodamine B rhodamine 123 rhodamine isothiocyanate Sulforodamine 101 sulfonyl chloride derivative (Texas Red) N,N,N',N'-tetramethyl-6-carboxyrodamine (TAMRA) Tetramethyl rhodamine tetramethyl rhodamine isothiocyanate (TRITC) Riboflavin Rosolic acid Metal chelates, for example, lanthanide chelates (e.g., europium terbium chelates), ruthenium chelates.

[0092] There is a large amount of practical guidance available in the literature for selecting pairs of Petition 870210110461, dated 11 / 29 / 2021, pp. 58 / 117 47 / 99 suitable donor-recipient matches for specific probes, as exemplified by the following references: Pesce et al., Eds, Fluorescence Spectroscopy (Marcel Dekker, New York, 1971); White et al., Fluorescence Analysis: A Practical Approach (Marcel Dekker, New York, 1970); and similar. The literature also includes references providing exhaustive lists of fluorescent and chromogenic molecules and their optical properties relevant to the choice of reporter-suppressor pairs (see, for example, Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd Edition (Academic Press, New York, 1971); Griffiths, Colour and Constitution of Organic Molecules (Academic Press, New York, 1976); Bishop, Ed., Indicators (Pergamon Press, Oxford, 1972); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, 1992); Pringsheim, Fluorescence and Phosphorescence (Interscience Publishers, New York, 1949); and similar).Furthermore, there is extensive guidance in the literature for the derivatization of reporter and suppressor molecules for covalent bonding through common reactive groups that can be added to a nucleic acid, as exemplified by the following references: Haugland (supra); Ullman et al., U.S. Patent No. 3,996,345; Khanna et al., U.S. Patent No. 4,351,760. Thus, it is well within the capabilities of those skilled in the art to select an energy exchange pair for a specific application and to conjugate the members of this pair to a probe molecule, such as, for example, a nucleic acid, peptide, or other polymer.

[0093] Generally, it is preferred that a band of Petition 870210110461, dated 11 / 29 / 2021, p. 59 / 117 48 / 99 Inhibitor absorption substantially overlaps the fluorescence emission band of the donor. When the donor (fluorophore) is a component of a probe that utilizes donor-acceptor energy transfer, the fluorescent donor portion and the suppressor (acceptor) of the invention are preferably chosen so that the donor and acceptor portions exhibit donor-acceptor energy transfer when the donor portion is excited. A factor to be considered in choosing the fluorophore-suppressor pair is the donor-acceptor energy transfer efficiency between them. Preferably, the FRET efficiency between the donor and acceptor portions is at least 10%, more preferably at least 50%, and even more preferably at least 80%. The FRET efficiency can be easily tested empirically using both methods described herein and known in the art.

[0094] The effectiveness of energy transfer between the donor-acceptor pair can also be adjusted by changing the ability of the donor and acceptor groups to dimerize or closely associate. If the donor and acceptor portions are known or determined to associate closely, an increase or decrease in association can be promoted by adjusting the length of a linking portion, or of the probe itself, between the donor and the acceptor. The ability of the donor-acceptor pair to associate can be increased or decreased by adjusting the hydrophobic or ionic interactions or steric repulsions in the probe construction. Thus, the intramolecular interactions responsible for the association of the donor-acceptor pair can be strengthened or weakened. Thus, for example, the association between the pair Petition 870210110461, dated 11 / 29 / 2021, pp. 60 / 117 The 49 / 99 donor-acceptor ratio can be increased, for example, by using a donor having an overall negative charge and an acceptor with an overall positive charge.

[0095] In addition to fluorophores, which are directly linked to a probe, fluorophores can also be linked indirectly. In this embodiment, a ligand molecule (e.g., biotin) is usually covalently linked to probe species. The ligand then binds to other molecules (e.g., streptavidin molecule), which is inherently detectable or covalently linked to a signaling system, such as a fluorescent compound, or an enzyme that produces a fluorescent compound by converting a non-fluorescent compound. Useful enzymes of interest as markers include, for example, hydrolases, particularly phosphatases, esterases and glycosidases, hydrolases, peptidases or oxidases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc., as discussed above.For a review of various marker or signal production systems that may be used, see U.S. Patent No. 4,391,904.

[0096] Donors for use in conjunction with the suppressors of the invention include, for example, xanthene dyes, including fluoresceins, cyanine dyes, and rhodamine dyes. Many suitable forms of these compounds are widely available commercially with substituents on their phenyl moieties, which can be used as the binding site, or as the binding functionality for binding to a nucleic acid. Petition 870210110461, dated 11 / 29 / 2021, pp. 61 / 117 50 / 99 Another group of fluorescent compounds used in conjunction with the suppressors of the invention are naphthylamines, with an amino group in the alpha or beta position. Included among these naphthylamino compounds are 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate, and 2-p-touidinyl-6-naphthalene sulfonate. Other donors include 3-phenyl-7-isocyanatocoumarin, acridines such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl)maleimide; benzoxadiazoles, stilbenes, pyrenes, and the like.

[0097] For the sake of clarity of illustration, the discussion that follows focuses on attaching suppressors and fluorophores to nucleic acids. The focus on nucleic acid probes is not intended to limit the scope of probe molecules to which suppressors can be attached. Those skilled in the art will appreciate that suppressors can also be attached to small molecules, proteins, synthetic peptides, solid carrier polymers, and the like, using standard synthetic chemistry.

[0098] In an exemplary embodiment, wherein the probe is a nucleic acid probe, the fluorophore is a Quasar® dye (Biosearch Technologies, Inc.). The fluorophore is preferably bound to either the 3' or 5' terminus of the nucleic acid, although internal sites are also accessible and useful for selected purposes. Whichever terminus the fluorophore is bound to, the inhibitor will generally be bound to its antipode, or to a position internal to the nucleic acid chain. Donor groups are preferably introduced using an amidite derivative of the donor. Alternatively, donor groups that Petition 870210110461, dated 11 / 29 / 2021, pp. 62 / 117 51 / 99 comprise reactive functional groups (e.g., isothiocyanates, active esters, etc.) that can be introduced by means of reaction with a reactive functional group in a lanyard or linker arm attached to the nucleic acid (e.g., hexylamine).

[0099] In yet another preferred embodiment, the donor moiety may be linked to the 3' terminal position of a nucleic acid through the use of a derivatized synthesis support. For example, TAMRA (tetramethylrhodamine carboxylic acid) is linked to a 3' terminal nucleic acid using a solid support that is derivatized with an analog of this fluorophore (Biosearch Technologies, Inc.)

[00100] In view of the well-developed body of literature relating to the conjugation of small nucleic acid molecules, many other methods for fixing the donor / acceptor pairs of nucleic acids will be apparent to those skilled in the art. For example, rhodamine and fluorescein dyes are conveniently linked to the 5' hydroxyl group of a nucleic acid at the conclusion of solid-phase synthesis by means of dyes derivatized with a phosphoramidite group (see, for example, Woo et al., U.S. Patent No. 5,231,191; and Hobbs, Jr., U.S. Patent No. 4,997,928).

[00101] More specifically, there are many binding moieties and methodologies for attaching 5' or 3' terminal groups of nucleic acids, as exemplified by the following references: Eckstein, editor, Nucleic Acids and Analogs: A Practical Approach (IRL Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (nucleic acid 3' thiol group); Sharma et al., Petition 870210110461, dated 11 / 29 / 2021, pp. 63 / 117 52 / 99 Nucleic Acids Research, 19:3019 (1991) (3'-sulfhydryl); Giusti et al., PCR Methods and Applications, 2:223-227 (1993) and Fung et al., U.S. Patent No. 4,757,141 (5'-phosphoamino group via Aminolink™ II available from PE Biosystems, CA); Stabinsky, U.S. Patent No. 4,739,044 (3-aminoalkylphosphoryl group); Agrawal et al., Tetrahedron Letters, 31:1543-1546 (1990) (attachment via phosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15:4837 (1987) (5-mercapto group); Nelson et al., Nucleic Acids Research, 17:7187-7194 (1989) (3'-amino group) and the like.

[00102] The methods for detecting fluorescent markers are well known to those skilled in the art. Thus, for example, fluorescent markers can be detected by exciting the fluorophore with the appropriate wavelength of light and detecting the resulting fluorescence. Fluorescence can be detected visually, using photographic film, or by using electronic detectors such as charge-coupled devices (CCDs) or photomultipliers, and similar devices. Similarly, enzyme markers can be detected by providing appropriate substrates to the enzyme and detecting the resulting reaction product. Reactive Functional Groups

[00103] The components of the compounds of the invention (for example, ligands, nucleoside, nucleotides, oligonucleotides, nucleic acid, carrier molecule, and the solid support) can be linked through binding sites formed by the reaction of a first and a second reactive functional group. The reactive functional groups are Petition 870210110461, dated 11 / 29 / 2021, pp. 64 / 117 53 / 99 of complementary reactivity, and react to form a covalent bond between two components of the compounds, referred to herein as a binding site. For example, compounds according to Formula (I) or (II), where Rx or Rs is a reactive functional group, can be reacted with a reactive functional group of complementary reactivity on the other component (such as a ligand, nucleoside, nucleotide, oligonucleotide, nucleic acid, carrier molecule, and solid support) to covalently link the components through the resulting binding site. The reactive functional group of complementary reactivity can be located at any position on the other component (ligand, nucleoside, etc.), for example, an alkyl or heteroalkyl group on an aryl or heteroaryl nucleus or a substituent on an aryl or heteroaryl nucleus.In several embodiments, when the reactive group is attached to an alkyl (or heteroalkyl) or substituted alkyl (or heteroalkyl) chain, the reactive group is preferably located at a terminal position of the chain.

[00104] The reactive groups and classes of reactions useful in the practice of the present invention are generally those well known in the art of bioconjugate chemistry. Currently, the preferred classes of reactions available with reactive precursors of the oligomers of the present invention are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to multiple carbon-carbon bonds and Petition 870210110461, dated 11 / 29 / 2021, pp. 65 / 117 54 / 99 carbon-heteroatom (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al., Modification of Proteins; Advances in Chemistry Series, vol. 198, American Chemical Society, Washington, DC, 1982.

[00105] By way of example, the reactive functional groups used in the present invention include, but are not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids, isonitrile, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, allenes, Ortho-esters, sulfites, enamines, inamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds. Reactive functional groups also include those used to prepare bioconjugates of, for example, hydroxysuccinimide, maleimide, and similar nesters.Methods for preparing each of these functional groups are well known in the art, and their application or modification for a particular purpose is within the capacity of a person skilled in the art (see, for example, Sandler and Karo, eds.). Petition 870210110461, dated 11 / 29 / 2021, pp. 66 / 117 55 / 99 Preparations of Organic Functional Groups, Academic Press, San Diego, 1989).

[00106] Useful reactive functional group conversions include, for example: (a) carboxyl groups, which are readily converted into different derivatives including, but not limited to, active esters (e.g., N-hydroxysuccinimide, N-hydroxybenzotriazole esters, thioesters, p-nitrophenyl esters), acid halides, acyl imidazoles, alkyl, alkenyl, alkynyl and aromatic esters; (b) hydroxyl groups, which can be converted into esters, ethers, halides, aldehydes, etc. (c) haloalkyl groups, wherein the halide can then be displaced with a nucleophilic group, such as, for example, an amine, a carboxylate anion, a thiol anion, a carbanion, or an alkoxide ion, thus resulting in the covalent bonding of a new group in place of the halogen atom; (d) dienophilic groups, which are capable of participating in Diels-Alder reactions, such as maleimide groups; (e) aldehyde or ketone groups, so that subsequent derivatization is possible through the formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or through mechanisms such as Grignard addition or alkyl-lithium addition; (f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides; (g) thiol groups, which can be, for example, Petition 870210110461, dated 11 / 29 / 2021, pp. 67 / 117 56 / 99 converted to disulfide or reacted with acyl halides; (h) amine or sulfhydryl groups, which may be, for example, acylated, alkylated or oxidized; (i) alkenes, which can be subjected to, for example, cycloadditions, acylation, Michael addition, etc.; (j) epoxides that can react with, for example, amines and hydroxyl compounds; and (k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis.

[00107] Reactive functional groups can be chosen in such a way that they do not participate in, or interfere with, the reactions necessary to assemble the oligomer of the invention. Alternatively, a reactive functional group can be protected from participating in the reaction by the presence of a protecting group. Those skilled in the art understand how to protect a particular functional group in such a way that it does not interfere with a set of chosen reaction conditions. For examples of useful protecting groups, see, for example, Greene et al., Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991. Covalent Bonding Portion

[00108] Included in some of the oligomers of the present invention is a reactive functional group portion capable of forming at least one covalent bond between the oligomer and a target sequence. Multiple covalent bonds can also be formed by providing a multiplicity of such units. The covalent bond is preferably a residue of Petition 870210110461, dated 11 / 29 / 2021, pp. 68 / 117 57 / 99 nucleobases in the target chain, but it can also be done with other parts of the target, including sugar or phosphodiester. The nature of the reaction of the crosslinking portion determines the nature of the target in the duplex. Preferred crosslinking portions include acylating and alkylating agents and, in particular, those positioned relative to the portion that confers sequence specificity so as to allow reaction with the target site in the chain.

[00109] The crosslinking portion can conveniently be placed as a pyrimidine analog or purine residue in the oligomer sequence. Placement can be at the 5' and / or 3' ends, the internal portions of the sequence, or combinations thereof. Placement at the ends to allow for greater flexibility is preferred. Analogous portions can also be linked to peptide backbones.

[00110] Examples of alkylation moieties that are useful in the invention include N4,N4-ethanecytosine and N6,N6-ethaneadenine.

[00111] Of course, the nucleobase need not be a purine or pyrimidine; in fact, the portion to which the reactive function is attached need not be a nucleobase at all and may be a sugar, a ligand, a suppressor, a stabilizing portion of a fluorophore, or some combination of these components of the oligomers of the invention. Any means of attaching the reactive group is satisfactory, provided the positioning is correct. Synthesis

[00112] The compounds of the invention (such as solid supports, monomers (e.g., phosphoramidites) and Petition 870210110461, dated 11 / 29 / 2021, pp. 69 / 117 58 / 99 oligomers of the present invention or segments thereof are generally conventionally synthesized. See, for example, U.S. Patent No. 7,019,129; U.S. Patent No. 8,466,266; and U.S. Patent No. 7,879,986. The synthetic methods known in the art and described herein can be used to synthesize oligomers containing the compounds of the invention, as well as other nucleobases known in the art, using appropriately protected nucleomonomers. Methods for the synthesis of oligomers are found, for example, in Froehler, B., et al., Nucleic Acids Res. (1986) 14:5399-5467; Nucleic Acids Res. (1988) 16:4831-4839; Nucleosides and Nucleotides (1987) 6:287-291; Froehler, B., Tetrahedron Letters (1986) 27:5575-5578; Caruthers, MH, in Oligodeoxynucleotide Inhibitions of Gene Expression (1989), JS Cohen, editor, CRC Press, Boca Raton, pages 7-24; Reese, CB et al., Tetrahedron Letters (1985) 28: 2245-2248. The synthesis of methylphosphonate-linked oligomers via the methylphosphoramidite chemical route has also been described (Agrawal, S. et al., Tetrahedron Letters (1987) 28:3539-3542; Klem, RE, et al., International Publication Number WO 92 / 07864).

[00113] An exemplary synthesis of several compounds of the invention is shown in Figure 2 and Example 1.

[00114] In an exemplary embodiment, nucleomonomers are directly incorporated into oligomers, or a fragment thereof, conveniently using standard synthesis conditions and reagents. Examples of linkages made by this method include phosphodiester, phosphorothioate, phosphoroamidate, methylphosphonate, phosphorodithioate, carbonate, Petition 870210110461, dated 11 / 29 / 2021, pp. 70-117 59 / 99 morpholino carbamate and sulfonate.

[00115] In several embodiments, the synthesis involves the synthesis of short synthons (dimers, trimers, etc.), starting with an appropriate precursor. This approach is suitable for the synthesis of linkages including N-methylhydroxylamine, dimethylhydrazone, sulfamate, carbamate, sulfonate, sulfonamide, formacetal, thioformacetal and carbonate.

[00116] The oligomers of the invention can be synthesized by any suitable chemistry including methods and conditions of coupling with amidite, triester or hydrogen phosphonate. The oligomers are preferably synthesized from suitable starting synthons, which are preferably protected at the 5' position with DMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PACO (phenoxyacetyl), a silyl ether such as TBDMS (t-butyldiphenylsilyl) or TMS (trimethylsilyl) and activated at the 3' end with an ester, H-phosphonate, an amidite such as β-cyanoethylphosphoramidite, a silyl ether such as TBDMS or TMS or t-butyldiphenyl. Alternatively, suitable uridine or cytidine precursors, such as blocked 5-iodo-2'-deoxyuridine, 5-iodo-2'-O-alkyluridine, 5-bromo-2'-deoxyuridine, 5-trifluoromethanesulfonate-2'-deoxyuridine, 5-bromo-2'-O-alkyluridine or blocked and protected 5-iodo-2'-deoxycytidine, 5-bromo-2'-deoxycytidine, 5-trifluoromethanesulfonate-2'-deoxycytidine, 5-iodo-2'-O-alkylcytidine, 5-bromo-2'-O-alkylcytidine may be conveniently incorporated into short oligomers, such as dimers, trimers, tetramers, Petition 870210110461, dated 11 / 29 / 2021, pp. 71 / 117 60 / 99 pentamers or longer synthons, which are subsequently derivatized to produce suitable synthons and longer oligomers.

[00117] Examples of oligomer synthesis containing about 4 or more nucleomonomer residues are carried out using synthons such as monomers, dimers or trimers carrying a coupling group suitable for use with amidite, H-phosphonate or triester chemistry. The synthon can be used to link the oligomer components via a phosphodiester bond or a phosphorus-containing non-phosphodiester bond (e.g., phosphorothioate, methylphosphonate, thionomethylphosphonate, phosphoramidate and the like).

[00118] The synthesis of other substituted ligands not containing phosphorus can be carried out using suitable precursors, as known in the art.

[00119] Once the desired nucleic acid is synthesized, it is preferably separated from the solid support on which it was synthesized and treated, by methods known in the art, to remove any protecting groups present (e.g., 60°C, 5h, concentrated ammonia). In these embodiments, where a base-sensitive group is attached to the nucleic acids (e.g., TAMRA), deprotection preferably utilizes milder conditions (e.g., butylamine:water 1:3, 8 hours, 70°C). Deprotection under these conditions is facilitated by the use of fast-deprotecting amides (e.g., dC-acetyl, dG-DMF).

[00120] Following cleavage from the scaffold and deprotection, the nucleic acid is purified by any Petition 870210110461, dated 11 / 29 / 2021, pp. 72 / 117 61 / 99 method known in the art, including chromatography, extraction and purification on gel. In a preferred embodiment, the nucleic acid is purified using HPLC. The concentration and purity of the nucleic acid isolate is preferably determined by measuring the optical density at 260 nm in a spectrophotometer. Oligomeric Tests and Probes of the Invention

[00121] In various embodiments, the present invention provides an oligomer for use in one or more test formats. In selected embodiments, the oligomer participates in the generation of a detectable signal by association or dissociation from its target. The oligomeric probes of the present invention are not limited in use to any particular test format. Therefore, the following description is intended to illustrate exemplary test formats in which the oligomers of the present invention find use, and is not intended to be limiting of the test formats in which the oligomers are used. Tests

[00122] The following discussion is generally relevant to the tests described herein. This discussion is intended to illustrate the invention by reference to certain preferred embodiments and should not be interpreted as limiting the scope of the probes and test types in which the compounds of the invention find use. Other test formats utilizing the compounds of the invention will be apparent to those skilled in the art.

[00123] In general, to determine the concentration of a target molecule, such as, for example, a nucleic acid of unknown quantity, it is preferable Petition 870210110461, dated 11 / 29 / 2021, pp. 73 / 117 62 / 99 first obtain reference data in which constant amounts of probe are contacted with nucleic acid standards that measure a range of known quantities. The fluorescence emission intensity of each of the reference mixtures is used to derive a standard graph or curve, in which the unknown concentration is compared with the intensity of the known standards. For example, a probe that: a) hybridizes with a sequence within the target nucleic acid; b) has fluorophore and suppressor modifications on the 5' and 3' termini being the labeling sites; and c) has fluorogenic character that is suppressed in an unbound conformation and then releases signal after binding to the target nucleic acid, can be used to obtain these reference data. Such a probe gives a characteristic fluorescence emission in which the signal increases as the concentration of the target nucleic acid increases.Next, a sample with an unknown target quantity is brought into contact with the probe, and the fluorescence intensity of the mixture is determined. The fluorescence emission intensity is then compared with reference standards to obtain the target concentration in the test mixture. Multiplex analysis

[00124] In another embodiment, the solid supports and oligomers of the present invention are used as a probe or a component of one or more probes used in a multiplex test for the detection of one or more species in a mixture.

[00125] Probes based on solid supports or Petition 870210110461, dated 11 / 29 / 2021, pp. 74 / 117 63 / 99 oligomers of the present invention are particularly useful in performing multiplex type analyses and tests. In a typical multiplex analysis, two or more different species (or regions of one or more species) are detected using two or more probes, wherein each probe is labeled with a different fluorophore. Preferred species used in multiplex analyses that rely on donor-acceptor energy transfer meet at least two criteria: the fluorescent species are bright and have a well-resolved spectrum; and the energy transfer between the fluorescent species and the quencher is effective.

[00126] The solid supports and oligomers of the present invention allow the design of multiplexed tests in which more than one fluorescent reporter is associated with one or more suppressor structures. A number of different multiplexed tests using the solid supports or oligomers of the invention will be apparent to one skilled in the art. In an exemplary test, each of at least two distinct fluorescent reporters is paired with the same type of suppressor structure on the respective oligomers, to modulate the FRET signal, either through contact or suppression. Alternatively, a test can be practiced in which at least two distinct fluorescent reporters are associated with distinct suppressor structures, so that the fluorescent properties are better combined. The fluorophores can be linked to the same molecule as the suppressor or to a different molecule. Furthermore, similar to the suppressor and the fluorophores, the molecules Petition 870210110461, dated 11 / 29 / 2021, pp. 75 / 117 64 / 99 carriers used in a particular test system, such as the oligo sequence that covalently links the fluorophore and suppressor, may be the same or different.

[00127] In addition to the mixtures described above, the present invention also provides a qualitative method for detecting the presence of particular molecular species. The method includes: (a) contacting the species with a mixture containing a solid support or oligomer of the invention; and (b) detecting a change in a fluorescent property of one or more components of the resulting mixture, thereby detecting the presence of the molecular species.

[00128] The simultaneous use of two or more probes, using donor-acceptor energy transfer, is known in the art. For example, multiplexed tests using nucleic acid probes with different sequence specificities have been described. Fluorescent probes have been used to determine whether an individual is homozygous wild-type, homozygous mutant, or heterozygous for a particular mutation. For example, using a fluorescein-suppressed molecular beacon that recognizes the wild-type sequence and another rhodamine-suppressed molecular beacon that recognizes a mutant allele, genotyping of individuals for the chemokine receptor is possible (β-Kostrikis et al., Science 279:1228-1229 (1998)). The presence of only a fluorescein signal indicates that the individual is wild-type, and the presence of only the rhodamine signal indicates that the individual is a homozygous mutant.The presence of both rhodamine and fluorescein signals is diagnostic of... Petition 870210110461, dated 11 / 29 / 2021, pp. 76 / 117 65 / 99 a heterozygote. Tyagi et al., Nature Biotechnology 16:4953 (1998) described the simultaneous use of four different labeled molecular beacons for allele discrimination, Lee et al., BioTechniques 27: 342-349 (1999) described homogeneous detection of seven colors of six PCR products.

[00129] The suppressors of the present invention can be used in multiplex tests designed to detect and / or quantify substantially any species, including, for example, whole cells, viruses, proteins (e.g., enzymes, antibodies, receptors), glycoproteins, lipoproteins, subcellular particles, organisms (e.g., Salmonella), nucleic acids (e.g., DNA, RNA and their analogues), polysaccharides, lipopolysaccharides, lipids, fatty acids, non-biological polymers and small molecules (e.g., toxins, drugs, pesticides, metabolites, hormones, alkaloids, steroids). Nucleic Acid Probes

[00130] The solid supports and oligomers of the present invention are useful nucleic acid probes and can be used as components of detection agents for a variety of DNA amplification / quantification strategies, including, for example, 5'-nuclease assay, Chain Displacement Amplification (SDA), Nucleic Acid Sequence-Based Amplification (NASBA), Rolling Circle Amplification (RCA), as well as for the direct detection of targets in solution-phase or solid-phase (e.g., matrix) assays. Furthermore, solid supports and oligomers can be used in probes for, Petition 870210110461, dated 11 / 29 / 2021, pp. 77 / 117 66 / 99 substantially any format, including, for example, selected molecular model formats, Scorpion™ probes, Sunrise™ probes, conformationally assisted probes, light-up probes, invader detection probes, and TaqMan™ probes. See, for example, Cardullo, R., et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988); Dexter, DL, J. Chem. Physics, 21:836-850 (1953); Hochstrasser, RA, et al., Biophysical Chemistry, 45: 33141 (1992); Selvin, P., Methods in Enzymology, 246:300-334 (1995); Steinberg, I., Ann. Rev. Biochem., 40:83-114 (1971); Stryer, I., Ann. Rev. Biochem., 47: 819-846 (1978); Wang, G., et al., Tetrahedron Letters, 31: 6493-6496 (1990); Wang, Y., et al., Anal. Chem., 67:1197-1203 (1995); Debouck, C., et al., In Supplement to Genetic Nature, 21:48-50 (1999); Rehman, FN, et al., Nucleic Acids Research, 27:649-655 (1999); Cooper, J.P., et al., Biochemistry, 29:9261-9268 (1990); Gibson, EM, et al., Genome Methods, 6:995-1001 (1996); Hochstrasser, RA, et al., Biophysical Chemistry, 45: 133-141 (1992); Netherlands, PM, et al., Proc. Natl. Acad. Sci. USA, 88:7276-7289 (1991); Lee, LG, et al., Nucleic Acids Research, 21:37613766 (1993); Livak, KJ, et al., PCR Methods and Applications, Cold Spring Harbor Press (1995); Vamosi, G., et al., Biophysical Journal, 71:972-994 (1996); widow, CT, et al., Biotechniques, 22: 176-181 (1997); widow, CT, et al., Biotechniques, 22: 130-38 (1997); Giesendorf, BAJ, et al., Clinical Chemistry, 44:482-486 (1998); Kostrikis, LG, et al., Science, 279:1228-1229 (1998); Matsuo, T., Biochemica et Biophysica Acta, 1379: 178-184 (1998); Piatek, AS, et al., Nature Biotechnology, 16:359Petição 870210110461, de 29 / 11 / 2021, pág. 78 / 117 67 / 99 363 (1998); Schofield, P., et al., Appl. Environ. Microbiology, 63: 1143-1147 (1997); Tyagi S., et al., Nature Biotechnology, 16:49-53 (1998); Tyagi, S., et al., Nature Biotechnology, 14:303-308 (1996); Nazarenko, L.A., et al., Nucleic Acids Research, 25: 2516-2521 (1997); Uehara, H., et al., Biotechniques, 26:552-558 (1999); Whitcombe D., et al., Nature Biotechnology, 17:804-807 (1999); Lyamichev, V., et al., Nature Biotechnology, 17:292 (1999); Daubendiek et al., Nature Biotechnology, 15:273-277 (1997); Lizardi, P.M., et al., Nature Genetics, 19:225-232 (1998); Walker, G., et al., Nucleic Acids Res., 20:16911696 (1992); Walker, G.T., et al., Clinical Chemistry, 42:9-13 (1996); e Compton, J., Nature, 350:91-92 (1991).

[00131] Thus, the present invention provides a method for detecting a target nucleic acid sequence. The method includes: (a) contacting the target nucleic acid sequence with a detector (for example, an oligomer of the present invention); (b) hybridizing the target-binding sequence with the target sequence, thereby altering the conformation of the nucleic acid detector, causing a change in a fluorescence parameter; and (c) detecting the change in the fluorescence parameter, thereby detecting the target nucleic acid sequence.

[00132] In the methods described herein, unless otherwise indicated, a preferred nucleic acid detector includes a single-stranded target-binding sequence. The binding sequence has attached to it: i) a fluorophore; and ii) a suppressor. The binding sequence also optionally has attached to it a stabilizing moiety. Furthermore, before its hybridization with a Petition 870210110461, dated 11 / 29 / 2021, pp. 79 / 117 68 / 99 complementary sequence, the detector nucleic acid is preferably in a conformation that allows donor-acceptor energy transfer between the fluorophore and the suppressor when the fluorophore is excited. Furthermore, in each of the methods described in this section, a change in fluorescence is detected as an indication of the presence of the target sequence. The change in fluorescence is preferably detected in real time.

[00133] Presently preferred nucleic acid probes do not require the nucleic acid to adopt a secondary structure for the probe to function. In this method, and unless otherwise indicated, the other methods described in this section, the detector nucleic acid may assume substantially any intramolecularly associated secondary structure, but this structure is preferably a member selected from hairpins, hairpin-loop structures, pseudo-knots, triple helices, and conformationally assisted structures. Furthermore, the intramolecular base-paired secondary structure preferably comprises a portion of the target-binding sequence.

[00134] In another aspect, the invention provides a method for detecting the amplification of a target sequence. The method involves the use of an amplification reaction, such as PCR. An exemplary amplification reaction includes one or more of the following steps: (a) hybridization of a nucleic acid sample comprising the target sequence of interest with PCR primers flanking the target sequence; (b) extension of hybridized primers with a Petition 870210110461, dated 11 / 29 / 2021, pp. 80-117 69 / 99 polymerase is used to produce the PCR product, and to separate the two strands of the PCR product to make the sense and antisense target sequence strands accessible; (c) hybridization of a nucleic acid detector of the sense or antisense strand of the target sequence in the PCR product, wherein the detector nucleic acid includes: i) a target single-stranded ligand sequence that is complementary to at least a portion of the sense or antisense strand of the target sequence in the PCR product and hybridizes with a region between the PCR primers; ii) a fluorophore; and iii) a suppressor of the present invention; In which, prior to its hybridization with the target sequence, the nucleic acid detector is in a conformation that allows donor-acceptor energy transfer between the fluorophore and the suppressor when the fluorophore is excited; altering, therefore, the conformation of the detector nucleic acid (e.g., linearizing any secondary structure or random helical conformations that contribute to quenching efficiency), causing a change in a fluorescence parameter (such as signal intensity); and (d) measuring the change in the fluorescence parameter to detect the target sequence and its amplification. Optionally, the variation in the fluorescence parameter can become permanent if the polymerase encounters the hybridized detector nucleic acid during primer extension (step (b) above) and hydrolyzes the oligomer that binds the fluorophore and the suppressor, such as by means of a Petition 870210110461, dated 11 / 29 / 2021, pp. 81 / 117 70 / 99 secondary nuclease polymerase activity.

[00135] In yet another aspect, the invention provides a method for determining a first nucleic acid and a second nucleic acid that hybridize. In this method, the first nucleic acid is an oligomer (in solution or bound to a solid support), according to the invention. The method includes: (a) contacting the first nucleic acid with the second nucleic acid; (b) detecting a change in a fluorescent property of a selected member from the first nucleic acid, the second nucleic acid, and a combination thereof, thereby determining whether hybridization occurs.

[00136] In various embodiments, the present invention provides probes and methods for use in detecting polymorphisms in target nucleic acid sequences. Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus where divergence occurs. Preferred markers have at least two alleles, each occurring with a frequency greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphic locus can be as small as a base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number tandem repeats (VNTRs), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, single sequence repeats, and insertion elements such as Alu. The first form Petition 870210110461, dated 11 / 29 / 2021, pp. 82 / 117 The identified allelic form 71 / 99 was arbitrarily designated as the reference form, and other allelic forms are designated as alternative or variant alleles. The allelic form that occurs most frequently in a selected population is often referred to as the wild-type form. Diploid organisms can be homozygous or heterozygous for allelic forms. A diallel polymorphism has two forms. A triallelic polymorphism has three forms.

[00137] In an exemplary embodiment, a probe of the invention is used to detect a single nucleotide polymorphism. A single nucleotide polymorphism occurs at a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is generally preceded by, and followed by, highly conserved sequences of the allele (e.g., sequences that vary from less than 1 / 100 or 1 / 1000 members of the populations). A single nucleotide polymorphism generally arises due to the substitution of one nucleotide for another at the polymorphic site. A transversion is the substitution of one purine for another purine or one pyrimidine for another pyrimidine. A transversion is the substitution of a purine for a pyrimidine or vice versa. Single nucleotide polymorphism can also arise from a nucleotide deletion or a nucleotide insertion relative to a reference allele.

[00138] An oligomer of the invention supporting both a suppressor and a fluorophore may be used or, alternatively, one or more of the nucleic acids may be individually labeled with a single member of a pair of Petition 870210110461, dated 11 / 29 / 2021, pp. 83 / 117 72 / 99 energy transfer (e.g., a suppressor or fluorophore). When a nucleic acid individually labeled with a suppressor is the probe, the interaction between the first and second nucleic acids can be detected by observing the interaction between the suppressor and the nucleic acid or, more preferably, the suppression by the suppressor of the fluorescence of a fluorophore bound to the second nucleic acid.

[00139] In some embodiments, a complex ground state between an inhibitor of the present invention and a fluorophore is formed. In an exemplary embodiment, both the suppressor and the fluorophore are conjugated with the same nucleic acid oligomer.

[00140] In addition to their general usefulness in probes designed to investigate nucleic acid amplification, polymorphism, and detection and quantification, the present oligomers and solid supports can be used in substantially any nucleic acid probe format now known or subsequently discovered. For example, the solid supports and oligomers of the present invention can be incorporated into probe portions, such as Taqman™ probes (Held et al., Genome Res. 6:986-994 (1996), Holland et al., Proc. Nat. Acad. Sci. USA 88:7276-7280 (1991), Lee et al., Nucleic Acids Res. 21:3761-3766 (1993)), molecular beacons (Tyagi et al., Nature Biotechnology 14:303-308 (1996), Jayasena et al., U.S. Patent No. 5,989,823, granted November 23, 1999)), Scorpion probes (Whitcomb et al., Nature Biotechnology 17:804-807 (1999)), sunrise probes (Nazarenko et al., Nucleic Acids Res. 25:2516-2521 (1997)), probes Petition 870210110461, dated 11 / 29 / 2021, pp. 84 / 117 73 / 99 conformationally assisted (Cook, R., US Patent Application co-pending and jointly assigned 2007 / 0059752, filed June 9, 1999), light probes based on peptide nucleic acids (ANPs) (Kubista et al., WO 97 / 45539, December 1997), double-strand-specific DNA dyes (Higuchi et al., Bio / Technology 10:413-417 (1992), Wittwer et al., BioTechniques 22:130-138 (1997)) and the like. These and other probe portions with which the present suppressors can be used are reviewed in Non-Isotopic DNA Probe Techniques, Academic Press, Inc. 1992.

[00141] The oligomers to be used in the probes of the invention may be of any suitable size, and are preferably in the range from about 2 to about 100 nucleotides, more preferably from about 10 to about 80 nucleotides, and even more preferably from about 10 to about 40 nucleotides. In doubly labeled probes (fluorophore suppressor), the donor moiety is preferably separated from the inhibitor by at least about 6, preferably at least about 8, preferably at least about 10 nucleotides, and more preferably by at least about 15 nucleotides. In various embodiments, the donor moiety is preferably linked to both the 3'- and 5'-terminal nucleotides of the probe. The suppressor portion is also preferentially bound to both the 3'- and 5'-terminal nucleotides of the probe.More preferably, the donor and acceptor portions are linked to the 3'- and 5'- or 5'- and 3'- terminal nucleotides of the probe, respectively, although the internal placement. Petition 870210110461, dated 11 / 29 / 2021, pp. 85 / 117 74 / 99 may also be useful.

[00142] The precise sequence and length of a nucleic acid probe of the invention depends, in part, on the nature of the target polynucleotide to which it binds. The binding site and length can be varied to achieve annealing and melting properties appropriate for the particular embodiment. Guidance for making such design choices can be found in many recognized references in the art.

[00143] In some embodiments, the 3'-terminal nucleotide of the nucleic acid probe is blocked or rendered incapable of extension by a nucleic acid polymerase. The blocking is conveniently effected by binding a donor or acceptor moiety to the 3'-terminal position of the nucleic acid probe, either directly or by a linker moiety.

[00144] The nucleic acid may comprise DNA, RNA, or mixtures or chimeric derivatives or modified versions thereof. Both the nucleic acid probe and target may be present as a single strand, duplex, triplex, etc. In addition, the nucleic acid may be modified in the nucleobase unit, sugar portion, or phosphate structure with other groups, such as radioactive markers, minor groove ligands, intercalating agents, acetylinically unsaturated hydrocarbons, fluoroalkyl groups, donor and / or acceptor groups, and the like.

[00145] The oligomers of the present invention are useful as primers that are discrete sequences or as primers with a random sequence. The primers of Petition 870210110461, dated 11 / 29 / 2021, pp. 86 / 117 75 / 99 random sequences are generally about 6 or 7 nucleomonomers in length. Such primers can be used in various nucleic acid amplification protocols (PCR, ligase chain reaction, etc.) or in cloning protocols. The 5' end substitutions of the present invention generally do not interfere with the oligomer's ability to function as a primer. Oligomers of the invention possessing 2' modifications at sites other than 3' terminal residue sites, other modifications that render the RNase H oligomer incompetent or otherwise nuclease-stable, can be advantageously used as probes or primers for RNA or DNA sequences in cell extracts or other solutions containing nucleases.Thus, oligomers can be used in protocols for nucleic acid amplification in a sample by mixing the oligomer with a sample containing target nucleic acid, followed by hybridization of the oligomer with the target nucleic acid and amplification of the target nucleic acid by PCR, LCR, or other suitable methods.

[00146] Oligomers derived with chelating agents, such as EDTA, DTPA, or 1,2-diaminocyclohexaneacetic acid analogs, can be used in various in vitro diagnostic tests as described (Patents Nos. 4,772,548, 4,707,440 and 4,707,352). Alternatively, the oligomers of the invention can be derivatized with crosslinking agents, such as 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine or 5-(3-(4-bromobutyramido)prop-1-yl)-2'-deoxyuridine and used in various test methods or kits as described (International Publication No. WO Petition 870210110461, dated 11 / 29 / 2021, pp. 87 / 117 76 / 99 90 / 14353).

[00147] In addition to the use described, the ability of oligomers to inhibit gene expression can be verified in in vitro systems by measuring expression levels in cells from individuals or in recombinant systems, using any suitable method (Graessmann, M., et al., Nucleic Acids Res. (1991) 19:53-59).

[00148] Conditions that favor hybridization between oligomer of the present invention and targeting nucleic acid molecules can be empirically determined by those skilled in the art, and may include optimal incubation temperatures, salt concentrations, length and compositions of nucleotide bases of analog oligonucleotide probes, and concentrations of oligomer and nucleic acid molecules in the sample. Preferably, hybridization is performed in the presence of at least one millimolar magnesium and at a pH greater than 6.0. In some embodiments, it may be necessary or desirable to treat a sample to render the nucleic acid molecules in the sample as single-stranded prior to hybridization. Examples of such treatments include, but are not limited to, treatment with a base (preferably followed by neutralization), incubation at elevated temperature, or treatment with nucleases.

[00149] Furthermore, because the salt dependence of nucleic acid hybridization is largely determined by the charge density of the backbone of an oligonucleotide hybridization analog, incorporating non-standard nucleotide analogs into the oligomer of the present invention may increase or decrease the salt dependence of Petition 870210110461, dated 11 / 29 / 2021, pp. 88 / 117 77 / 99 Hybridization. This modulation can be used to advantage in the methods of the present invention, where it may, in some respects, be desirable to be able to increase the rigor of hybridization by altering the salt conditions, for example, or to release a hybridized nucleic acid by reducing the salt concentration. In still other aspects of the present invention, it may be desirable to have high-affinity binding of an oligonucleotide analog of the present invention to a nucleic acid at very low salt content. In this case, positioning nucleotide monomers with uncharged backbone portions to an oligonucleotide of the present invention is advantageous.

[00150] The high degree of specificity of the oligomers of the present invention in binding to nucleic acid target molecules allows the practitioner to select hybridization conditions that may favor discrimination between nucleic acid sequences comprising a sequence portion that is completely complementary to at least a portion of one or more oligomers and nucleic acid target molecules comprising a sequence portion comprising a small number of non-complementary nucleobases within a substantially complementary sequence.For example, hybridization or washing temperatures can be selected in such a way as to allow stable hybrids between the oligomer of the present invention and nucleic acid target molecules that are fully complementary along a sequence stretch, but promote the dissociation of hybrids between the oligomer of the present invention and nucleic acid target molecules that are not fully complementary, including those that... Petition 870210110461, dated 11 / 29 / 2021, pp. 89 / 117 78 / 99 involve one or two unpairings of nucleotide bases along a complementary sequence segment. The selection of a hybridization temperature and washes may depend, at least in part, on other conditions, such as salt concentration, the concentration of the oligomer and nucleic acid target molecules, the relative proportions of oligomer to nucleic acid target molecules, the lengths of the oligomers to be hybridized, the nucleobase composition of the oligomer and nucleic acid target molecules, the monomer composition of the oligonucleotide analog molecules, etc.Furthermore, when selecting for conditions that favor stable hybrids of completely complementary molecules and disfavor stable hybrids between oligomers and nucleic acid target molecules that are incompatible by one or more nucleobases, additional conditions may be taken into consideration and, whenever desirable, altered, including but not limited to, the length of the oligonucleotide analog to be hybridized, the length of the complementarity sequence portion between the oligomer and nucleic acid target molecules, the number of non-complementary nucleotide bases within a complementarity sequence portion, the identity of non-matching nucleotide bases, the identity of nucleobases in the vicinity of unpaired nucleobases, and the relative position of any unpaired nucleobases along a complementarity stretch.Technicians in the field of nucleic acid hybridization would be able to determine favorable hybridization and washing conditions using the present oligomer. Petition 870210110461, dated 11 / 29 / 2021, pp. 90 / 117 79 / 99 invention for the hybridization of nucleic acid target molecules, depending on the particular application. Favorable conditions may be those that favor stable hybrids between oligomer and nucleic acid target molecules that are, at least in part, substantially complementary, including those comprising one or more mismatches.

[00151] Favorable conditions may be those that favor stable hybrids between oligomers and nucleic acid target molecules that are, at least in part, completely complementary and disfavor or destabilize hybrids between molecules that are not completely complementary.

[00152] Using methods such as those described herein, the melting temperature of the oligomer of the present invention hybridizes to target nucleic acid molecules of different sequences can be determined and can be used in determining favorable conditions for a given application. It is also possible to empirically determine favorable hybridization conditions, for example, hybridization of target nucleic acid molecules to oligomers that are attached to a solid support and detection of hybridized complexes.

[00153] The target nucleic acid molecules that are bound to solid supports or oligomeric probes of the present invention can be conveniently and efficiently separated from unbound nucleic acid molecules in the research population by direct or indirect binding of oligomer probes to a solid support. A solid support can be washed in Petition 870210110461, dated 11 / 29 / 2021, pp. 91 / 117 80 / 99 highly stringent conditions for removing nucleic acid molecules that are not bound to oligomer probes. However, the binding of oligomer probes to a solid support is not a requirement of the present invention. For example, in some applications bound and unbound nucleic acid molecules can be separated by centrifugation through a matrix or by phase separation or, in some cases, by other forms of separation (e.g., differential precipitation) which may optionally be aided by chemical groups incorporated into the oligomer probes (see, for example, Patent No. 6,060,242 granted on May 9, 2000, to Nie et al.). Nucleic Acid Capture Probes

[00154] In one embodiment, an immobilized nucleic acid comprising an inhibitor is used as a capture probe. The immobilized nucleic acid optionally further comprises a stabilizing portion. The nucleic acid probe can be directly linked to a solid support, for example, by 3'- or 5'- nucleotide terminal linkage of the probe to the solid support. More preferably, however, the probe is linked to the solid support by a linking element (supra). The linker serves to distance the probe from the solid support. The linker is most preferably about 5 to about 30 atoms in length, more preferably about 10 to about 50 atoms in length.

[00155] In several embodiments, the solid support is also used as a synthesis support in the preparation of the oligomer (probe). The length and chemical stability of the linker between the solid support and the first 3' unit Petition 870210110461, dated 11 / 29 / 2021, pp. 92-117 The 81 / 99 nucleic acid linker arm plays an important role in the efficient synthesis and hybridization of support-linked nucleic acids. The linker arm is preferably long enough so that a high yield (>97%) can be achieved during automated synthesis. The required linker length will depend on the particular solid support used. For example, a six-atom linker is generally sufficient to achieve a yield >97% during automated nucleic acid synthesis when highly cross-linked polystyrene is used as a solid support. The linker arm is preferably at least 20 atoms long in order to achieve a high yield (>97%) during automated synthesis when CPG is used as a solid support.

[00156] Hybridization of a probe immobilized on a solid support generally requires that the probe be separated from the solid support by at least 30 atoms, more preferably by at least 50 atoms. In order to achieve this separation, the linker usually includes a spacer positioned between the linker and the 3' terminus. For nucleic acid synthesis, the linker arm is normally linked to the 3'-OH of the 3' terminus by an ester bond that can be cleaved with basic reagents to release the nucleic acid from the solid support.

[00157] A wide variety of ligands that can be used to attach the nucleic acid probe to the solid support are known in the art. The ligand can be formed from any compound that does not significantly interfere with the hybridization of the target sequence with the probe attached to the solid support. The ligand can be formed Petition 870210110461, dated 11 / 29 / 2021, pp. 93-117 82 / 99, for example, a nucleic acid homopolymer, which can be easily added to the ligand by automated synthesis. Alternatively, polymers, such as functionalized polyethylene glycol, can be used as a ligand. Such polymers are currently preferred over homopolymeric nucleic acids because they do not significantly interfere with the hybridization of the probe with the target nucleic acid. Polyethylene glycol is particularly preferred because it is commercially available, soluble in both organic and aqueous media, easy to functionalize, and completely stable under nucleic acid synthesis and post-synthesis conditions.

[00158] The linkage sites between the solid support, the ligand, and the probe are preferably not cleaved during synthesis or removal of nucleobase protecting groups under basic conditions at elevated temperature. These linkages may, however, be selected from groups that are cleavable under a variety of conditions. Examples of currently preferred linkages include carbamate, ester, and amide linkages. Detection of Nucleic Acids in Samples

[00159] The solid supports and oligomers of the present invention can be used for nucleic acid detection. Such detection methods include: providing a sample, contacting at least one oligonucleotide analog of the present invention with the sample under conditions that permit hybridization of the oligomer with nucleic acid molecules, and detecting one or more nucleic acid molecules from the sample that have been hybridized with one or more oligomers of the present invention. Petition 870210110461, dated 11 / 29 / 2021, pp. 94 / 117 83 / 99

[00160] The sample can be from any source, and can be a biological sample, such as a sample of an organism or a group of organisms from the same or different species. The biological sample can be a sample of body fluid, for example, a blood sample, serum sample, lymph sample, bone marrow sample, ascitic fluid, pleural fluid, pelvic lavage fluid, ocular fluid, urine, semen, sputum, or saliva. The biological sample can also be a skin, nasal, throat, or genital swab, or extracts of fecal material. Biological samples can also be samples of organs or tissues, including tumors. Biological samples can also be samples of cell cultures, including both cell lines and primary cultures of prokaryotic and eukaryotic cells.

[00161] The sample may be from the environment, such as a body of water or soil, or from food, drink, or a water source, an industrial source, workplace, public area, or living room. A sample may be an extract, for example, a liquid extract from a soil sample or foodstuffs. A sample may be a solution made by washing or soaking, or a suspension of a swab from articles such as tools, clothing, artifacts, or other materials.

[00162] A sample can be a transformation or a processed sample; processing can involve steps that increase the purity, concentration, or accessibility of the sample components to facilitate sample analysis. As non-limiting examples, processing can include Petition 870210110461, dated 11 / 29 / 2021, pp. 95 / 117 84 / 99 steps that reduce the volume of a sample, remove or separate components of a sample, solubilize a sample or one or more components of the sample, or interrupt, modify, expose, release or isolate components of a sample. Non-limiting examples of such procedures are centrifugation, precipitation, filtration, homogenization, cell lysis, antibody binding, cell separation, etc. For example, in some preferred embodiments of the present invention, the sample is a blood sample that is at least partially processed, for example, by removing red blood cells, by concentration, by selecting one or more types of cells or viruses (e.g., white blood cells or pathogenic cells), or by lysis of the cells, etc.

[00163] Exemplary samples include a solution of nucleic acid molecules that have been at least partially purified. The nucleic acid molecules may be from a single source or multiple sources and may comprise DNA, RNA, or both. For example, a solution of nucleic acid molecules may be a sample that has been subjected to any of the steps of cell lysis, concentration, extraction, precipitation, nucleic acid selection (such as, for example, poly-A RNA selection or selection of DNA sequences comprising Alu elements), or treatment with one or more enzymes. The sample may also be a solution comprising synthetic nucleic acid molecules.

[00164] An oligomer or solid support of the present invention may be any oligomer form disclosed herein, or any oligomer comprising a monomer, dimer or Petition 870210110461, dated 11 / 29 / 2021, pp. 96-117 85 / 99 component that is not a nucleic acid (e.g., linker, fluorophore, suppressor, stabilizing moiety) disclosed herein. An oligonucleotide analog used in the methods of the present invention may be of any length and any composition of nucleobases, and may comprise one or more radicals of nucleic acids, peptides, proteins, lipids, carbohydrates, steroids, and other chemical and biochemical moieties. An oligonucleotide analog of the present invention may be provided in solution or bound to a solid support. In some preferred embodiments of the present invention, the oligomer comprises non-standard nucleotide analogs.

[00165] Methods for detecting bound nucleic acids are well known in the art, and may include the use of a detectable marker that is bound to, or incorporated into, nucleic acid molecules of the study population or that binds to, or is incorporated into, a hybridized target nucleic acid molecule or hybridized target nucleic acid molecule complex. Detectable markers for nucleic acid molecules are well known in the art, and comprise fluorescent molecules such as fluorophores (including those set forth herein), radioisotopes, bulk-altered chemical groups, specific binding members such as biotin, which can be detected by signal-generating molecules and the like.Detectable markers can also be incorporated into, or linked to, the oligomer of the present invention, for example, in cases where sandwich hybridization using a signal oligomer is used for detection or detection is performed using a specific linking member, such as a... Petition 870210110461, dated 11 / 29 / 2021, pp. 97 / 117 86 / 99 antibody that recognizes oligomer / target nucleic acid molecule complexes. Solid supports can be scanned, exposed to a film, visually inspected, etc., to determine the presence of a detectable marker and thus determine the binding of a target nucleic acid molecule to an oligomer immobilized on a solid support, such as those of the present invention. Kits

[00166] One aspect of the present invention is the formulation of kits that facilitate the practice of syntheses using the compounds of the invention (such as solid supports of the invention or monomers of the present invention) and tests using the oligomers of the invention, as described above. The kits of the invention typically comprise a compound of the invention (such as a solid support of the invention, or an oligomer of the present invention), either present as a chemically reactive species useful for the preparation of conjugates, or present as a terminated oligomer, wherein the oligomer is a member of a specific bonding pair. The kit optionally further comprises one or more buffering agents, typically present as an aqueous solution.The kits of the invention also optionally comprise additional detection reagents, a purification medium for the purification of the resulting labeled substance, luminescence standards, enzymes, enzyme inhibitors, organic solvents, or instructions for performing a test of the invention. Other kit formats will be apparent to those skilled in the art and are within the scope of the present invention. Petition 870210110461, dated 11 / 29 / 2021, pp. 98 / 117 87 / 99

[00167] In summary, in exemplary embodiments, the present invention provides: A compound having a structure according to Formula I or II: regardless of R1a and R1b, together with the carbon atom to which they are attached, are optionally joined to form a ring that is a member selected from substituted or unsubstituted C3-C7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl. R7a and R7b, together with the carbon atom to which they are attached, are optionally joined to form a ring that is a member selected from substituted or unsubstituted C3-C7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl. R7a and R7b, together with the carbon atom to which they are attached, are optionally joined to form a ring that is a member selected from substituted or unsubstituted C3-C7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl. At least one of R1a, R1b, R7a and R7b is not H. R8R is selected from H, * and ™. Lx is Petition 870210110461, dated 11 / 29 / 2021, pp. 99 / 117 88 / 99 is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl. Lx is selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl. R is selected from a protected or unprotected functional reactive group, a bonding site, and a solid support. Rx is selected from a protected or unprotected functional reactive group and a bonding site.Each binding site is covalently linked to a member independently selected from a nucleoside, a nucleoside ligand, a nucleotide, a nucleotide ligand, an oligonucleotide, an oligonucleotide ligand, a nucleic acid, a nucleic acid ligand, a carrier molecule, a carrier molecule ligand, a solid support, and a solid support ligand.

[00168] A compound according to the preceding paragraph, wherein R1 and R1b are independently selected from unsubstituted C1, C2, C3, C4, C5 and Cg alkyl groups.

[00169] A compound according to the previous paragraph, wherein R1 and R1b are each methyl.

[00170] A compound according to any preceding paragraph, wherein R7 and R7b are each H.

[00171] A compound according to any preceding paragraph, in which R7 and R7b are, independently, Petition 870210110461, dated 11 / 29 / 2021, pp. 100 / 117 89 / 99 selected from unsubstituted C1, C2, C3, C4, C5 and C1C alkyl.

[00172] A compound according to any preceding paragraph, in which R7 and R7b are each methyl.

[00173] A compound according to any preceding paragraph, wherein R1 and R1b are each H.

[00174] A compound according to any preceding paragraph, wherein R1a, R1b, R7a and R7b are independently selected from unsubstituted C1, C2, C3, C4, C5 and C6 alkyl groups.

[00175] A compound according to any preceding paragraph, wherein R1a, R1b, R7a and R7b are each methyl. [0017e] A compound according to any preceding paragraph, wherein Lx is selected from C1, C2, C3, C4, C5, C6, C7, C8, C9 and C10 not substituted.

[00177] A compound according to any preceding paragraph, in which Lxsé is substituted heteroalkyl.

[00178] A compound according to any preceding paragraph, wherein Rx is selected from a phosphoramidite, -OH, -ODMT, -COOH, an active ester and -NH2.

[00179] A compound according to any preceding paragraph, wherein R is selected from -OH, -ODMT and a bonding site covalently linked to a ligand on a solid support.

[00180] A compound according to any preceding paragraph, wherein Rx is selected from a phosphoramidite, -OH, -ODMT, -COOH, an active ester and -NH2.

[00181] A compound according to any preceding paragraph, wherein Rx is a binding site covalently linked to a nucleoside ligand, wherein the Petition 870210110461, dated 11 / 29 / 2021, pp. 101 / 117 90 / 99 nucleoside has the following structure: The ring marked B is a nucleobase. Rn3e-OH or a phosphoramidite. Rn5e-OH or -ODMT.

[00182] A compound according to any previous paragraph, where R8 is selected from: OOM' NCtCHzJjCrP-NPrj iOHE)5

[00183] A method for detecting a target nucleic acid sequence, the method comprising: (a) contacting the target sequence with a nucleic acid detector; comprising a single-stranded target-binding sequence, the nucleic acid detector having bound to it i) a fluorophore; and ii) a compound according to any paragraph. Petition 870210110461, dated 11 / 29 / 2021, pp. 102 / 117 91 / 99 previous, where R8 comprises a binding site covalently linked (directly or through a ligand) to the nucleic acid detector; in which the nucleic acid detector is in a conformation that allows the transfer of donor-acceptor energy between the fluorophore and the compound when the fluorophore is excited; (b) hybridize the target-binding sequence with the target sequence, thereby altering the conformation of the detector nucleic acid, causing a change in a fluorescence parameter; and (c) detect the change in the fluorescence parameter, thereby detecting the target nucleic acid sequence.

[00184] A method for determining whether a first nucleic acid and a second nucleic acid hybridize, the first nucleic acid comprising a compound according to any preceding paragraph, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to the first nucleic acid, the method comprising: (a) to contact the first nucleic acid with the second nucleic acid; and (b) to detect a change in a fluorescent property of a selected member from among the first nucleic acid, the second nucleic acid, and a combination thereof, thereby determining whether hybridization occurs.

[00185] A method for monitoring a nucleic acid amplification reaction, the method comprising: (a) preparation of an amplification reaction mixture comprising a nucleic acid detector having it Petition 870210110461, dated 11 / 29 / 2021, pp. 103 / 117 92 / 99 connected, i) a fluorophore; and ii) a compound according to any preceding paragraph, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to the nucleic acid detector; (b) subjecting the amplification reaction mixture to amplification conditions; (c) monitoring the reaction mixture for a fluorescent signal from the nucleic acid detector to obtain a test result; and (d) using the test result to monitor the nucleic acid amplification reaction.

[00186] A method for detecting the amplification of a target sequence, the method comprising: (a) hybridization of a nucleic acid sample comprising the target sequence with PCR primers flanking the target sequence; (b) extension of the hybridized primers with a polymerase to produce the PCR product, and separating the two strands of the PCR product to make the sense and antisense target sequence strands accessible; (c) hybridization of a nucleic acid detector of the sense or antisense strand of the target sequence in the PCR product, wherein the detector nucleic acid includes: i) a single-stranded target-binding sequence that is complementary to at least a portion of the sense or antisense strand of the target sequence in the PCR product and hybridizes with a region between the PCR primers; Petition 870210110461, dated 11 / 29 / 2021, pp. 104 / 117 93 / 99 ii) a fluorophore; and iii) a compound according to any preceding paragraph, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to the nucleic acid detector; In which, prior to its hybridization with the target sequence, the nucleic acid detector is in a conformation that allows donor-acceptor energy transfer between the fluorophore and the compound when the fluorophore is excited; (d) altering the conformation of the detector nucleic acid, thus causing a change in a fluorescence parameter; and (d) measuring the change in the fluorescence parameter to detect the target sequence and its amplification.

[00187] The materials and methods of the present invention are further illustrated by the following examples. These examples are offered to illustrate, but not to limit, the claimed invention. EXAMPLES Example 1: Synthesis of examples of Compounds of the Invention

[00188] The synthesis of several exemplary compounds of the invention is outlined in Figure 2.

[00189] Hydroxytetramethyljulolidine 2 was prepared from hydroxyaniline 1 without the need for chromatography, with an overall yield of approximately 50% for the two steps (Dyes and Pigments, 2003, 59, 63). Increasing the school yielded 95 g of 2 with relative ease. A test reaction of 2 with Fast Black K (FBK) salt yielded the deep blue dye 3. Conversion of 2 to the Cghydroxyl compound 4 was performed using excess of Petition 870210110461, dated 11 / 29 / 2021, pp. 105 / 117 94 / 99 chlorohexanol and potassium carbonate in DMF, under anhydrous conditions. The literature process for alkylation using sodium hydride in dry DMF was avoided to minimize difficulties in large-scale production. The addition of a methanol-salt water solution of Fast Black K to a methanol solution of 4 provided azo dye alcohol 5, which was converted to amidite 6. Scale-up yielded approximately 13 g of amidite. The amidite was used to make 5'-labeled poly T oligonucleotides, which were purified by double HPLC and then used to determine the extinction coefficients for the dye.

[00190] The reaction of bromohexanoic acid with 2, as above, does not yield compound 7. The desired bromohexanoic acid was converted to its methyl ester 8, which was then coupled to 2 to obtain the respective C6-ester compound, which was not isolated. Cleavage of the methyl ester using 2 M aqueous sulfuric acid in THF gave the C6-acid compound 7. Coupling to the FBK salt gave the azo-acid dye 10, which was used to prepare the DMT-protected serinol derivative 11. This was sequentially converted to glycolate 12 and then to CPG 13. CPG was used to make the 3'-labeled poly T oligos, which were purified by double HPLC, and then used to determine the quenching coefficients for the dye. As with 5'-labeled oligonucleotides, no significant traces of dye decomposition or byproducts are seen by HPLC in crude oligonucleotide samples after deprotection under Petition 870210110461, dated 11 / 29 / 2021, pp. 106 / 117 95 / 99 basic conditions. 0 azo-acid dye 10 was converted into the active ester 14. Example 2: Performance Comparison of BHQ2, BBQ, and Cosmic Suppressor Introduction

[00191] The performance of a Cosmic Suppressor (shown below) was evaluated for quenching dyes that emit light at longer wavelengths. Specifically, the inhibitor's ability to quench signals from the fluorophores Quasar 670 and Quasar 705 was examined. For reference, the performance of BHQ2 and BlackBerry suppressors (BBQ-650®; Berry & Associates, Inc.; described in U.S. Patent No. 7,879,986) was also examined when paired with these same fluorophores. TaqMan probes containing each of these suppressors were evaluated using real-time PCR and digestion tests. BBQ-B5Ü Diagram: Portions of Suppressors Petition 870210110461, dated 11 / 29 / 2021, pp. 107 / 117 96 / 99 Methods

[00192] A TaqMan model assay was used for all comparisons performed in this study, and designed to detect the human gene for muscarinic cholinergic receptors 3 (Gene ID CHRM3, accession number nm_000740). The probe sequence is 5'-[Fluorophore]TCCTTTGGGCTCCTGCCATCT-[Suppressor]-3' and signals the amplification product of the PCR primers 5'TTGGGTCATCTCCTTTG-3' and 5'-GCACAGTTCTCTTTCCA-3'. This probe sequence was synthesized with each of five different combinations of suppressor fluorophores: Quasar 670-Cosmic Suppressor, Quasar 670-BHQ2, Quasar 670BBQ, Quasar 705-Cosmic Suppressor, and Quasar 705-BHQ2. Each probe was associated with the same set of primers, and their inhibition efficiencies were evaluated using triplicate PCR amplification reactions from 25 ng of human genomic DNA. The composition of each reaction is as follows: Component (Standard Solution) Volume Final Concentration Nuclease-free water 10.25 pL N / A Platinum Taq PCR buffer (10X) 5.00 pL 1X TaqMan probe (10 pM) 2.00 pL 100 nM MgCl2 (50 mM) 2.00 pL 2.0 mM dNTPs (2.5 mM each) 4.00 pL 200 pM each Human genomic DNA (150 pg / pL) 25.0 pL 3.75 ng per reaction Platinum Taq polymerase (1 U / pL) 0.25 pL 1.25 units per reaction Forward primer (10 pM) 1.50 pL 300 nM Reverse primer (10 pM) Total Volume: 1.50 pL : 50.0 pL 300 nM Petition 870210110461, dated 11 / 29 / 2021, pages 108 / 117 97 / 99 Real-time PCR reactions were run on a RotorGene 6000 (Corbett Research), with Quasar 670 detected in the red channel (625 nm source, 660 nm detector, gain = 5) and Quasar 705 detected in the crimson channel (680 nm source, 710 nm detector, gain = 10). The 5 probes were then examined by a nuclease digestion assay in which 1.5 units of micrococcal nuclease are added to a 100 μL solution of nuclease buffer with a probe at a concentration of 400 nM. Digestion is allowed to proceed for a period of 10 minutes. After this interval, the signal intensity is measured on a Tecan Safire fluorimeter with the following settings: Quasar 670 and Quasar 705 configurations Measurement mode: Top fluorescence Top fluorescence Wavelength 641 nm 680 nm excitation: Wavelength 676 nm 715 nm emission: Bandwidth 12 nm 12 nm excitation: Bandwidth 12 nm 12 nm emission: Gain: 150 150 Number of flashes: 20 20 Delay time: 0 ps 0 ps Integration time: 20 ps 20 ps Signal-to-noise ratios are calculated by dividing the fluorescence intensity of the probe in the nuclease-treated reaction by the intensity of the same probe in an untreated reaction, after first subtracting the signal from Petition 870210110461, dated 11 / 29 / 2021, pp. 109 / 117 98 / 99 white buffer each. Real-Time PCR Results

[00193] Within the Quasar 670 graphs (Figure 3), the solid amplification traces represent the Cosmic Suppressor probe, the dashed traces represent the BlackBerry Suppressor (BBQ) probe, and the dotted traces represent the BHQ2 probe. Within the Quasar 705 graphs (Figure 4), the solid traces denote the Cosmic Suppressor probe, while the dotted traces denote the BHQ2 probe. With any fluorophore, Cosmic Suppressor demonstrates more efficient suppression than BHQ2 or BBQ, as evidenced by the baseline signal strength before the start of amplification. Nuclease Digestion Results

[00194] The nuclease test reveals that the two fluorophores were suppressed more efficiently by Cosmic Suppressors, in particular with Quasar 705 which shows 1 / 6 of the background fluorescence compared to BHQ2. Due to its superior quenching efficiency, Cosmic Suppressor shows the highest signal-to-noise ratio with Quasar 705 and the second highest with Quasar 670.

[00195] Figure 5A shows the fluorescence intensity measured with and without digestion of probes labeled with Cosmic Suppressor Quasar 670, Quasar 670-BHQ2, Quasar 670-BBQ, Cosmic Suppressor Quasar 705, and Quasar 705-BHQ2 in a nuclease assay. Figure 5B shows the signal-to-noise ratios calculated from the digested and undigested fluorescence of probes labeled with Cosmic Suppressor Quasar 670, Quasar 670-BHQ2, Quasar 670-BBQ, Cosmic Suppressor Quasar 705, and Quasar 705-BHQ2 in a digestion assay. Petition 870210110461, dated 11 / 29 / 2021, pages 110 / 117 99 / 99 nuclease.

[00196] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or alterations in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and scope of this application and are considered within the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Petition 870210110461, dated 11 / 29 / 2021, pp. 111 / 117

Claims

1 / 6 CLAIMS 1. Compound characterized by having a structure according to Formula I or II: wherein R1a, R1b, R7a and R7b are selected independently from C1, C2, C3, C4, C5 and C6 unsubstituted alkyl, and RH HL*-R' p R* · R8 is selected from H, ' > and ' where Lx is selected from a substituted or unsubstituted C1-C24 alkyl bond, substituted or unsubstituted C1-C24 cycloalkyl bond, substituted or unsubstituted C1-C24 heteroalkyl bond comprising at least one heteroatom selected from the group consisting of B, O, N, Si and S, and substituted or unsubstituted C1-C24 heterocycloalkyl bond comprising at least one heteroatom selected from the group consisting of B, O, N, Si and S;Lxs is selected from substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C1-C24 cycloalkyl, substituted or unsubstituted C1-C24 heteroalkyl comprising at least one heteroatom selected from the group consisting of B, O, N, Si and S, and substituted or unsubstituted C1-C24 heterocycloalkyl Petition 870210110461, 29 / 11 / 2021, p. 112 / 117 2 / 6 comprising at least one heteroatom selected from the group consisting of B, O, N, Si and S; Rs is selected from a protected or unprotected functional reactive group, a binding site, and a solid support; and RX is selected from a protected or unprotected functional reactive group and a binding site;wherein each binding site is covalently linked to a member independently selected from a nucleoside, a nucleoside ligand, a nucleotide, a nucleotide ligand, an oligonucleotide, an oligonucleotide ligand, a nucleic acid, a nucleic acid ligand, a carrier molecule, a carrier molecule ligand, a solid support, and a solid support ligand.

2. Compound according to claim 1, characterized in that R1a and R1b are each methyl.

3. Compound according to claim 1 or 2, characterized in that R7a and R7b are each methyl.

4. Compound according to any one of claims 1 to 3, characterized in that Lxs is a substituted heteroalkyl comprising at least one heteroatom selected from the group consisting of B, O, N, Si and S.

5. A compound according to any one of claims 1 to 4, characterized in that Rs are selected from -OH, -ODMT and a binding site covalently bonded to a ligand on a solid support.

6. Compound, according to any of the Petition 870210110461, dated 11 / 29 / 2021, pp. 113 / 117 3 / 6 claims 1 to 3, characterized by Lx being selected from unsubstituted C1, C2, C3, C4, C5, C6, C7, C8, C9 and C10 alkyl compounds.

7. Compound according to any one of claims 1 to 6, characterized by Rx being selected from a phosphoramidite, -OH, -ODMT, -COOH, an active ester and -NH2.

8. A compound according to any one of claims 1 to 6, characterized in that Rx is a binding site covalently linked to a nucleoside ligand, wherein said nucleoside has the structure: Rrt3 in which the ring marked B is a nucleobase; Rn3 is -OH or a phosphoramidite; and Rn5 is -OH or -ODMT.

9. Compound, according to any one of claims 1 to 3, characterized in that R8 is selected from: ϊΐίE NC(CH7bOΡ'NPΡΓϊ . and NC(CH2)5O''P''NPr; Petition 870210110461, dated 11 / 29 / 2021, pp. 114 / 117 4 / 6 10. Method for detecting a nucleic acid target sequence characterized by comprising: (a) bringing said target sequence into contact with a nucleic acid detector; comprising a single-stranded target-binding sequence, said nucleic acid detector having attached to it, i) a fluorophore; and ii) a compound, as defined in any one of claims 1 to 9, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to said nucleic acid detector; wherein said nucleic acid detector is in a conformation that permits donor-acceptor energy transfer between said fluorophore and said compound when said fluorophore is excited; (b) hybridizing said target-binding sequence to said target sequence, thereby altering said conformation of said nucleic acid detector, causing a change in a fluorescence parameter;and (c) detect said change in said fluorescence parameter, thereby detecting said target nucleic acid sequence.

11. Method for determining whether a first nucleic acid and a second nucleic acid hybridize, said first nucleic acid comprising a compound, as defined in any one of claims 1 to 9, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to said first nucleic acid, said method characterized by comprising: (a) contacting said first nucleic acid with said second nucleic acid; and (b) detecting a change in a fluorescent property of a member selected from said first nucleic acid, said second nucleic acid and a combination thereof, thereby determining whether said hybridization occurs.

12. Method for monitoring a nucleic acid amplification reaction characterized by comprising: (a) preparing an amplification reaction mixture comprising a nucleic acid detector having attached thereto i) a fluorophore; and ii) a compound, as defined in any one of claims 1 to 9, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to said nucleic acid detector; (b) subjecting the amplification reaction mixture to amplification conditions; (c) monitoring the reaction mixture for a fluorescent signal from the nucleic acid detector to obtain a assay result; and (d) employing the assay result to monitor the nucleic acid amplification reaction.

13. Method for detecting the amplification of a target sequence characterized by comprising: (a) hybridizing a nucleic acid sample Petition 870210110461, 11 / 29 / 2021, p. 116 / 117 6 / 6 comprising the target sequence with PCR primers flanking the target sequence; (b) extending the hybridized primers with a polymerase to produce the PCR product, and separating the two strands of the PCR product to make accessible the sense and presense strands of the target sequence; (c) hybridizing a sense or presense strand detector nucleic acid of the target sequence into the PCR product, wherein the detector nucleic acid comprises: i) a single-stranded target-binding sequence that is complementary to at least a portion of the sense or presense strand of the target sequence in the PCR product and hybridizes with a region between the PCR primers; ii) a fluorophore;and iii) a compound, as defined in any one of claims 1 to 9, wherein R8 comprises a binding site covalently linked (directly or through a ligand) to said nucleic acid detector; wherein prior to its hybridization with the target sequence, the nucleic acid detector is in a conformation that allows donor-acceptor energy transfer between the fluorophore and the compound when the fluorophore is excited; thereby altering the conformation of the nucleic acid detector, causing a change in a fluorescence parameter; and (d) measuring the change in the fluorescence parameter to detect the target sequence and its amplification. Petition 870210110461, dated 11 / 29 / 2021, p. 117 / 117;

Images