Left-handed gamma-peptide nucleic acids, methods of synthesis and uses therefor
10851407 ยท 2020-12-01
Assignee
Inventors
- Danith H. Ly (Pittsburgh, PA, US)
- Wei-Che Hsieh (Pittsburgh, PA, US)
- Iulia Sacui (Owensboro, KY, US)
- Arunava Manna (Pittsburgh, PA, US)
Cpc classification
International classification
C07K14/00
CHEMISTRY; METALLURGY
Abstract
A method of making optically pure preparations of chiral PNA (gamma peptide nucleic acid) monomers is provided. Nano structures comprising chiral PNA structures also are provided. Methods of amplifying and detecting specific nucleic acids, including in situ methods are provided as well as compositions and kits useful in those methods. Lastly, methods of converting nucleobase sequences from right-handed helical PNA, nucleic acid and nucleic acid analog structures to left-handed PNA, and vice-versa, are provided.
Claims
1. A method of amplifying a target nucleic acid sequence, comprising: a) contacting a target nucleic acid with a composition comprising: i) a sensor comprising: (1) a protecting (P) strand of achiral peptide nucleic acid and/or right-handed gamma-peptide nucleic acid (RyPNA) having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction, a first section having nucleobase sequence n, second section having nucleobase sequence m comprising unnatural nucleobases, third section having nucleobase sequence a and fourth section having nucleobase sequence b and comprising an N-terminal cysteine, sulfhydryl or protected sulfhydryl group, and a thioester bond linking sections m and n; (2) a sensing (S) strand of RyPNA or achiral peptide nucleic acid (achiral PNA) having an N-terminal end and a C-terminal end, hybridized to the P strand, comprising, in an N-terminal to C-terminal direction a first section having nucleobase sequence b complementary to sequence b, a second section having nucleobase sequence a complementary to sequence a, a third section having nucleobase sequence n complementary to sequence n and a fourth, toe-hold section having nucleobase sequence o, the S strand having a sequence complementary to a nucleotide sequence of a nucleic acid and comprising the target sequence, having a 5 end and a 3 end, comprising, in a 5 to 3 direction, without intervening nucleobases a first section having nucleobase sequence 0 complementary to sequence o, a second section having nucleobase sequence N complementary to sequence n, a third section having nucleobase sequence A complementary to sequence a and a fourth section having nucleobase sequence B complementary to sequence b, wherein hybridization of the target nucleic acid to the S strand displaces the P strand, and the first section of the displaced P strand having nucleobase sequence n is removed by self-splicing; ii) a converter comprising: (1) an achiral peptide nucleic acid (achiral PNA) having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction, a first section having nucleobase sequence b complementary to sequence b, a second section having nucleobase sequence a complementary to sequence a, and a third section having nucleobase sequence m having unnatural nucleobases complementary to sequence m; and (2) a left-handed PNA (LPNA) having an N-terminal end and a C-terminal end, hybridized to the achiral PNA, comprising, in an N-terminal to C-terminal direction a first section having nucleobase sequence b complementary to sequence b of the PNA, and a second section having nucleobase sequence a complementary to sequence a of the PNA, wherein the displaced P strand hybridizes to the achiral PNA of the converter, displacing the L PNA from the converter; and b) detecting the displaced L PNA, wherein, other than where sequences are indicated as being complementary, the sequences A, a, a, B, b, b, N, n, n, 0, o, m or m are not complementary to each other.
2. The method of claim 1, in which the displaced LPNA is detected by a cascading hybridization chain reaction.
3. The method of claim 2, in which the composition further comprises: iii) a first amplifier of LPNA having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction a first section having nucleobase sequence b, a second section having nucleobase sequence c, a third section having nucleobase sequence b and a fourth section having nucleobase sequence a; and iii) a second amplifier of LPNA having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction, a first section having nucleobase sequence c, a second section having nucleobase sequence b, a third section having nucleobase sequence a and a fourth section having nucleobase sequence b, wherein sequences of c and c do not hybridize to sequences a, a, b or b.
4. The method of claim 3, wherein the first amplifier comprises a first member of a Fluorescent Resonance Energy Transfer (FRET) pair, an excimer pair, a fluorescence dequenching pair, or a fluorescence activation pair, and the second amplifier comprises a second member of the FRET pair, the excimer pair, the fluorescence dequenching pair, or the fluorescence activation pair, respectively.
5. The method of claim 1, wherein each of section a, b, n and o consists of from 2 to 5 bases, and m consists of from 3 to 7 bases.
6. The method of claim 1, wherein the P strand is from 14 to 21 bases in length.
7. The method of claim 1, wherein the first section of the P strand is achiral PNA and the second, third and fourth sections of the P strand is RPNA.
8. The method of claim 1, wherein the unnatural nucleobases are selected from the group consisting of isoguanine, isocytosine, xanthine, diaminopyridine; 2-amino-6-dimethylaminopurine, 2-oxopyridine, 6-amino-5-nitro-3-(1- -D-2-deoxyribofuranosyl)-2(1H)-pyridone (dZ), 2-amino-8-(1--D-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP), ##STR00009##
9. A composition for use in detection of a target nucleic acid, comprising: a) a sensor comprising: i) a protecting (P) strand of achiral peptide nucleic acid and/or right-handed gamma-peptide nucleic acid (RPNA) having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction, a first section having nucleobase sequence n, second section having nucleobase sequence m comprising unnatural nucleobases, third section having nucleobase sequence a and fourth section having nucleobase sequence b and comprising an N-terminal cysteine, sulfhydryl or protected sulfhydryl group, and a thioester bond linking sections m and n; ii) a sensing (S) strand of RPNA or achiral peptide nucleic acid (achiral PNA) having an N-terminal end and a C-terminal end, hybridized to the P strand, comprising, in an N-terminal to C-terminal direction a first section having nucleobase sequence b complementary to sequence b, a second section having nucleobase sequence a complementary to sequence a, a third section having nucleobase sequence n complementary to sequence n and a fourth, toe-hold section having nucleobase sequence o, the S strand having a sequence complementary to a nucleotide sequence of a nucleic acid and comprising the target sequence, having a 5 end and a 3 end, comprising, in a 5 to 3 direction, without intervening nucleobases a first section having nucleobase sequence 0 complementary to sequence o, a second section having nucleobase sequence N complementary to sequence n, a third section having nucleobase sequence A complementary to sequence a and a fourth section having nucleobase sequence B complementary to sequence b, wherein hybridization of the target nucleic acid to the S strand displaces the P strand, and the first section of the displaced P strand having nucleobase sequence n is removed by self-splicing; b) a converter comprising, i) an achiral peptide nucleic acid (achiral PNA) having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction, a first section having nucleobase sequence b complementary to sequence b, a second section having nucleobase sequence a complementary to sequence a, and a third section having nucleobase sequence m having unnatural nucleobases complementary to sequence m; and ii) a left-handed PNA (LPNA) having an N-terminal end and a C-terminal end, hybridized to the achiral PNA, comprising, in an N-terminal to C-terminal direction a first section having nucleobase sequence b complementary to sequence b of the PNA, and a second section having nucleobase sequence a complementary to sequence a of the PNA, wherein the displaced P strand hybridizes to the achiral PNA of the converter, displacing the LPNA from the converter; and wherein, other than where sequences are indicated as being complementary, the sequences A, a, a, B, b, b, N, n, n, 0, o, m or m are not complementary to each other.
10. The composition of claim 9, further comprising: c) a first amplifier of LPNA having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction without intervening nucleobases, a first section having nucleobase sequence b, a second section having nucleobase sequence c, a third section having nucleobase sequence b and a fourth section having nucleobase sequence a; and d) a second amplifier of LPNA having an N-terminal end and a C-terminal end, comprising, in an N-terminal to C-terminal direction without intervening nucleobases, a first section having nucleobase sequence c, a second section having nucleobase sequence b, a third section having nucleobase sequence a and a fourth section having nucleobase sequence b, wherein sequences of c and c do not hybridize to sequences a, a, b or b.
11. The composition of claim 10, wherein the first amplifier comprises a first member of a Fluorescent Resonance Energy Transfer (FRET) pair, an excimer pair, a fluorescence dequenching pair, or a fluorescence activation pair, and the second amplifier comprises a second member of the FRET pair, the excimer pair, the fluorescence dequenching pair, or the fluorescence activation pair, respectively.
12. The composition of claim 9, each of section a, b, n and o consisting of from 2 to 5 bases, and m consisting of from 3 to 7 bases.
13. The composition of claim 9, wherein the unnatural nucleobases are selected from the group consisting of isoguanine, isocytosine, xanthine, diaminopyridine; 2-amino-6-dimethylaminopurine, 2-oxopyridine, 6-amino-5-nitro-3-(1- -D-2-deoxyribofuranosyl)-2 (1H)-pyridone (dZ), 2-amino-8-(1--D-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4 (8H)-one (dP), ##STR00010##
14. A method of detecting a nucleic acid comprising a specific nucleic acid sequence in a sample, comprising: a) hybridizing the nucleic acid to a duplex right-handed and/or achiral gamma-peptide nucleic acid (PNA) comprising a protecting (P) strand comprising, in order a first section comprising the specific nucleic acid sequence, a second section comprising a sequence of unnatural nucleobases, and a third section comprising a sequence of the nucleic acid adjacent to the sequence of the first section, and further comprising a thioester bond between the second and third sections and an N-terminal sulfhydryl group, hybridized to a sensing (S) strand comprising, in order a sequence complementary to the first section and the third section, but not the second section, such that the P strand comprising the specific nucleic acid sequence is displaced by the nucleic acid, and the third section of the P strand is removed by self-splicing between the sulfydryl and thioester; b) converting the P strand to an LPNA by hybridizing the P strand to a duplex converter PNA comprising a first strand of an achiral PNA having a sequence complementary to the P strand hybridized to a second strand of an LPNA having the same sequence as the first section of the P strand, thereby displacing the LPNA; and c) amplifying the displaced LPNA by concatenization of a pair of single-stranded LPNA amplifiers having a hairpin structure, the first amplifier having, in order, a sequence complementary to the displaced LPNA, a sequence that is not present in the displaced LPNA, and a portion of (not the complete sequence) the sequence of the displaced LPNA such that the hairpin of the first amplifier is opened by binding to the displaced LPNA, leaving a single-stranded tail comprising the sequence not present in the displaced LPNA and the portion of the sequence of the displaced LPNA, the second amplifier comprising a hairpin structure comprising, in order, a first portion having a sequence complementary to the sequence of the single-stranded tail formed by the binding of the LPNA to the first amplifier and a second portion having the sequence of the LPNA such that binding of the second amplifier to the single-stranded tail formed by the first amplifier binding to the LPNA opens the hairpin of the second amplifier, leaving a single-stranded tail having the same sequence as the LPNA, so that the first amplifier can bind to the single stranded tail resulting from binding of the second amplifier to the single-stranded tail formed by the first amplifier binding to the LPNA.
15. The method of claim 14, wherein the first amplifier comprises a first member of a Fluorescent Resonance Energy Transfer (FRET) pair, an excimer pair, a fluorescence dequenching pair, or a fluorescence activation pair, and the second amplifier comprises a second member of the FRET pair, the excimer pair, the fluorescence dequenching pair, or the fluorescence activation pair, respectively.
16. The method of claim 14, wherein the sample is a cell, cell lysate or tissue.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(27) The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges are both preceded by the word about. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, unless indicated otherwise, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values. As used herein a and an refer to one or more.
(28) As used herein, the term comprising is open-ended and may be synonymous with including, containing, or characterized by. The term consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. The term consisting of excludes any element, step, or ingredient not specified in the claim. As used herein, embodiments comprising one or more stated elements or steps also include, but are not limited to embodiments consisting essentially of and consisting of these stated elements or steps.
(29) The methods and compositions described herein are capable of many variations in detailed implementation, which may be derived from the description contained herein by a person of ordinary skill in the art.
(30) As used herein, the term nucleic acid refers to deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Nucleic acid analogs include, for example and without limitation: 2-O-methyl-substituted RNA, locked nucleic acids, unlocked nucleic acids, triazole-linked DNA, peptide nucleic acids, morpholino oligomers, dideoxynucleotide oligomers, glycol nucleic acids, threose nucleic acids and combinations thereof including, optionally ribonucleotide or deoxyribonucleotide residue(s). An oligonucleotide may be referred to by the length (i.e. number of nucleotides) of the strand, through the nomenclature -mer. For example, an oligonucleotide of 22 nucleotides would be referred to as a 22-mer. The peptide nucleic acids, nucleic acids and analogs thereof, comprise a nucleobase sequence and can bind specifically to each other with certain limitations as shown in
(31) In a first aspect of the disclosure, a method of transferring sequence information of a nucleic acid, nucleic acid analog or right-handed PNA (RPNA) to a left-handed PNA (LPNA) or from an LPNA to a nucleic acid, nucleic acid analog or RPNA, is provided. Also provided is a method of converting a target nucleobase sequence of a nucleic acid, nucleic acid analog or RPNA to a LPNA, or converting a target nucleobase sequence of an LPNA to a nucleic acid, nucleic acid analog or RPNA. As shown in
(32) In another aspect, as indicated above, LPNA, RPNA, nucleic acids and nucleic acid analogs all can hybridize to achiral PNA. As such, nanostructures are provided that comprise at least achiral PNA hybridized to LPNA and/or RPNA. Other nucleic acids or nucleic acid analogs can be included in the nanostructure, such as a structure comprising achiral PNA, LPNA and one or more of a nucleic acid or a nucleic acid analog. In one embodiment, the achiral PNA is hybridized to RPNA and not (substantially) to LPNA, and in another embodiment, the achiral PNA is hybridized to LPNA and not (substantially) to RPNA, recognizing the synthesis methods provided herein that permit production of optically purer preparations of RPNA and LPNA monomers.
(33) Nucleic acid analogs are broadly-known and are polymers having a sequence of nucleobases and a backbone. Nucleic acid analogs, in the context of all aspects of the disclosure presented herein, can hybridize to achiral PNA, RPNA, LPNA, RNA, or DNA by Watson-Crick base pairing, as with A binding T/U or C binding G, or Watson-Crick-like base-pairing, as in the case of unnatural nucleobases, such as isoC binding isoG. Non-limiting examples of common nucleic acid analogs include peptide nucleic acids, such as PNA, phosphorothioate (e.g.,
(34) Sequences of nucleic acids are written in their 5 to 3 orientation. Complementarity refers to a relationship between two structures that follow a simple lock-and-key principle. Complementarity is a property shared between two nucleic acid sequences, such that when aligned, the nucleotide bases at each position in the sequences are complementary, i.e. they are able to bind to, or hybridize with one another. Complementarity, also refers to the concept of purine nucleotide bases complementing, or being able to bind to and/or hybridize with, pyrimidine nucleotide bases. For example, complementarity in DNA and RNA occur between A and T, or A and U, and C and G (i.e., A-T/U, T/U-A, G-C or C-G). This concept of complementarity between nucleotide bases is referred to and known in the art as Watson-Crick base pairing. Reverse complementation refers to the idea that two sequences are complementary to one another when aligned in an antiparallel manner. The two sequences are in a sense, mirror images of one another. Antiparallel refers to the concept of two biopolymers, for example DNA or RNA, having opposite alignments. In DNA and RNA, opposite alignment refers to the 5-phosphoryl end of one sequence being aligned to the 3-hydroxyl end of another sequence. When two strands of nucleic acid are hybridized, they do so by reverse complementation, that is, a first strand in a 5 to 3 orientation hybridizes to a strand in its 3 to 5 orientation. Thus, the sequence 5-ATGC-3 hybridizes to a nucleic acid having the sequence: 5-GCAT-3, and are considered to be complementary. Sequences of nucleic acid analogs, such as PNAs are expressed in an orientation-specific manner as the 5 to 3 convention with respect to nucleic acids. Thus an achiral PNA or RPNA having the sequence N.sub.term-GCAT-C.sub.term will hybridize to the nucleic acid having the reverse-complementary sequence 5-ATGC-3, and thus are considered complementary. When two strands or nucleic acids, achiral PNA and PNA are said to be hybridized, it is meant that they are hybridized in water, normal saline, PBS, isotonic or other aqueous solutions with similar salt concentrations (e.g., 5%, 10%, 15% or 20%) at a temperature in the range of 25 C. to 50 C., for example at 45 C., 40 C., 37 C., 35 C., 30 C. or room temperature (e.g., 21 C.5 C.). For any kit, composition or reaction as described herein that is to be transported and used in a variety or ambient temperatures, such as outdoors in hot weather, it is preferred that the duplexed and hairpin reagents remain in their duplexed or hairpin states until they are displaced or converted by introduction of a target nucleic acid sequence.
(35) In terms of the nucleic acids, peptide nucleic acids, and left-handed and right-handed PNAs described herein, strands or portions thereof that hybridize together are complementary and are in reverse-complementary orientation to each other, as in Watson-Crick base pairing. Sections and sequences of each nucleic acid, peptide nucleic acid or -peptide nucleic acid are described as being in a particular sequential order, and a complementary nucleic acid or peptide nucleic acid (generally, referred to as a complementary sequence) has the respective complementary sections and sequences thereof in complementary orientation. As an example, as shown in
(36) ##STR00001##
(37) Likewise, strands or portions thereof that have the same nucleobase sequence, and the same orientation of sections, even though they may have different backbones (such as nucleic acid, achiral PNA, or right- or left-handed PNA), are said to have the same orientation. Thus, in
(38) The methods described herein are applicable to any specific nucleotide sequence, the specific hybridization of which according to standard Watson-Crick base pairing to a complementary sequence is expected to apply to any sequence of nucleobases, and thus one of ordinary skill would recognize that listing the of the specific nucleobase sequence of any of the sections would be unnecessary and superfluous. Thus any section of any length described herein comprises a sequence of any permutation of the nucleobases A, G, C and T/U or an unnatural nucleobase, for each nucleobase. Thus, the target nucleic acid can comprise any sequence of nucleobases, and includes a sequence (5 to 3) of sections O-N-A-B, which can be any useful sequence, the methods described herein are expected to operate correctly for any such useful sequence. That said, in a given genome, transcriptome and/or exome, it is most desirable to select a sequence for the target sequence that presents a unique sequence in the genome, transcriptome and/or exome to ensure the accuracy of the described assay.
(39) A PNA monomer, such as an achiral PNA monomer, an RPNA monomer or an LPNA monomer, incorporated into a PNA oligomer or polymer, is referred to herein as a PNA monomer residue, with each residue having the same or different nucleobase, such as adenine, guanine, cytosine, thymine and uracil bases, unnatural nucleobases, or other nucleobases, such that the order of bases on the PNA is its sequence, as with DNA or RNA. The PNA, nucleic acid or nucleic acid analog monomers and residue structures illustrated herein show a backbone monomer or a backbone monomer residue, respectively, attached to a nucleobase (R). A sequence of nucleobases in a PNA, nucleic acid or a nucleic acid analog oligomer or polymer, such as a PNA oligomer or polymer, binds to a complementary sequence of adenine, guanine, cytosine, thymine and/or uracil residues in a nucleic acid strand by cooperative bonding, essentially as with Watson-Crick binding of complementary bases in double-stranded DNA or RNA. Watson-Crick-like bonding refers to hydrogen bonding of nucleobases other than G, A, T, C or U, such as the bonding of the unnatural bases shown herein with each other.
(40) A peptide nucleic acid refers to a DNA or RNA mimic in which the sugar phosphodiester backbone of the DNA or RNA is replaced by a N-(2-aminoethyl)glycine unit. A gamma PNA (PNA) is an oligomer or polymer of gamma-modified N-(2-aminoethyl)glycine monomers of the following structure:
(41) ##STR00002##
where at least one of R1 or R2 attached to the gamma carbon is not a hydrogen, such that the gamma carbon is a chiral center. When R1 and R2 are hydrogen (N-(2-aminoethyl)-glycine backbone), there is no such chirality about the gamma carbon, and the resultant PNA is achiral PNA, often referred to in the art simply as PNA. An incorporated achiral PNA or PNA monomer,
(42) ##STR00003##
is referred to herein as an achiral PNA or PNA residue, with each residue having the same or different R group as its base (nucleobase), such as adenine, guanine, cytosine, thymine and uracil bases, or other bases, such as the unnatural (e.g., orthogonal) bases described herein, such that the order of bases on the PNA is its sequence, as with DNA or RNA. A sequence of nucleobases in a nucleic acid or a nucleic acid analog oligomer or polymer, such as an achiral PNA or PNA oligomer or polymers, binds to a complementary sequence of adenine, guanine, cytosine, thymine, uracil or unnatural residues in a nucleic acid or nucleic acid analog strand by nucleobase pairing, essentially as with double-stranded DNA or RNA. The following depict residue structures for achiral PNA (1), Left-handed PNA (2) and right-handed PNA (3).
(43) ##STR00004##
R1s each independently are selected from the group consisting of H (for achiral PNA), an amino acid side chain, a substituted amino acid sidechain, linear or branched (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, (C.sub.1-C.sub.8)hydroxyalkyl, (C.sub.1-C.sub.8)thiolalkyl, (C.sub.1-C.sub.8) aminoalky, (C.sub.1-C.sub.8) azidoalkyl, (C.sub.3-C.sub.8)aryl, (C.sub.3-C.sub.8)cycloalkyl, (C.sub.3-C.sub.8)aryl(C.sub.1-C.sub.6)alkylene, (C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkylene, CH.sub.2(OCH.sub.2CH.sub.2).sub.qOP.sub.1, CH.sub.2(OCH.sub.2CH.sub.2).sub.qNHP.sub.1, CH.sub.2(OCH.sub.2CH.sub.2-0).sub.qSP.sub.1, CH.sub.2(SCH.sub.2CH.sub.2).sub.qSP.sub.1, CH.sub.2(OCH.sub.2CH.sub.2).sub.rOH,CH.sub.2(OCH.sub.2CH.sub.2).sub.rN.sub.3, CH.sub.2(OCH.sub.2CH.sub.2).sub.rNH.sub.2, CH.sub.2(OCH.sub.2CH.sub.2), NHC(NH)NH.sub.2, or CH.sub.2(OCH.sub.2CH.sub.2).sub.rSS[CH.sub.2CH.sub.2].sub.8NHC(NH)NH.sub.2, where P1 is selected from the group consisting of H, (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, (C.sub.3-C.sub.8)aryl, (C.sub.3-C.sub.8)cycloalkyl, (C.sub.3-C.sub.8)aryl(C.sub.1-C.sub.6)alkylene, (C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkylene, substituted (C.sub.1-C.sub.8)alkyl, substituted (C.sub.2-C.sub.8)alkenyl, substituted (C.sub.2-C.sub.8)alkynyl, substituted (C.sub.1-C.sub.8)hydroxyalkyl, substituted (C.sub.3-C.sub.8)aryl, substituted (C.sub.3-C.sub.8)cycloalkyl, substituted (C.sub.3-C.sub.8)aryl(C.sub.1-C.sub.6)alkylene, substituted (C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkylene, substituted CH.sub.2(OCH.sub.2CH.sub.2).sub.qOP.sub.1, substituted CH.sub.2(OCH.sub.2CH.sub.2).sub.qNHP.sub.1, substituted CH.sub.2(OCH.sub.2CH.sub.2-0).sub.qSP.sub.1, substituted CH.sub.2(SCH.sub.2CH.sub.2).sub.qSP.sub.1, substituted CH.sub.2(OCH.sub.2CH.sub.2).sub.rOH, substituted CH.sub.2(OCH.sub.2CH.sub.2).sub.rNH.sub.2, substituted CH.sub.2(OCH.sub.2CH.sub.2).sub.rNHC(NH)NH.sub.2, or substituted CH.sub.2(OCH.sub.2CH.sub.2).sub.rSS[CH.sub.2CH.sub.2].sub.8NHC(NH)NH.sub.2, where P1 is selected from the group consisting of H, (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, (C.sub.3-C.sub.8)aryl, (C.sub.3-C.sub.8)cycloalkyl, (C.sub.3-C.sub.8)aryl(C.sub.1-C.sub.6)alkylene, (C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkylene; q is an integer from 0 to 50, inclusive, r and s are each independently integers from 1 to 50, inclusive, a polyether, a diether, an amino acid, and a polypeptide of from 2 to 25 amino acid residues, wherein the substituted moieties are substituted with one or more halo (e.g., F, Cl or Br), linear or branched (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, (C.sub.1-C.sub.8)hydroxyalkyl, (C.sub.3-C.sub.8)aryl, (C.sub.3-C.sub.8)cycloalkyl, amino acid side chain, amino acid, oligopeptide of from 2 to 25 residues, or hereroatom (e.g., S, O, N or P). By substituted amino acid side chain, it is meant an amino acid sidechain, such as a substituted lysine, arginine, serine, or cysteine sidechain, that is substituted with a group, such as an amino acid or oligopeptide, a (C.sub.1-C.sub.8) hydrocarbyl group, or, with any group listed above for R1. According to one aspect the substituted amino acid side chain is not substituted with a second substituted amino acid side chain, or alternately, the substituted amino acid side chain is not substituted with a second substituted amino acid side chain substitute with a further substituted amino acid side chain.
(44) Certain embodiments of the P strand of the sensor has the following alternate structures, with either a sulthydryl group (e.g., as with a cysteine) or a disulfide group at its N-terminal or C-terminal.
(45) ##STR00005##
where each instance of R is, independently a nucleobase that either forms hydrogen bonds with nucleobases of natural nucleic acids (e.g., A, T/U, G, or C), or unnatural nucleic acids. Values for n and m are each, independently from 1 to 25, 1-20, 5-10, and any increment therebetween. In the structure P of the sensor, the sequence n falls between the terminal sulthydryl and the internal thioester, with the sequence m of unnatural nucleobases being immediately adjacent to the internal thioester opposite to the sequence n. R1 is as described above. R2 is one or more amino acid residues, an amino acid side chain, linear, branched or hetero-substituted (C.sub.1-C.sub.8)alkyl, (C.sub.2-C.sub.8)alkenyl, (C.sub.2-C.sub.8)alkynyl, (C.sub.1-C.sub.8)hydroxyalkyl, (C.sub.3-C.sub.8)aryl, (C.sub.3-C.sub.8)cycloalkyl, (C.sub.3-C.sub.8)aryl(C.sub.1-C.sub.6)alkylene, or (C.sub.3-C.sub.8)cycloalkyl(C.sub.1-C.sub.6)alkylene. R3 is OH or NH.sub.2. Formulas (4), (5), (6) and (7) each depict an N-terminal Cysteine providing the N-terminal sulfhydryl, with formulas (6) and (7) depicting R1 as being H for the n sequence of the described P strand, affording additional backbone flexibility of achiral PNA, as compared to RPNA when R1 is not H, for the self-splicing restorator step in which the n sequence of P is removed. R3 is OH or NH.sub.2.
(46) A nucleobase is a nitrogenous base of a nucleotide. Natural nucleobases are adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Natural nucleobases include modified versions of A, G, C, T and U, but still bind to (are complementary to or hybridize to) one of A, G, C, T and U. An unnatural nucleobase used herein synonymously with an orthogonal nucleobase is a nucleobase that does not hybridize, or hybridizes unfavorably to one of the natural nucleobases: A, G, C or T/U. Unnatural nucleobases can hybridize (base-pair) to other unnatural nucleobases. As used herein, portions of the PNA reagents include unnatural or orthogonal nucleobase sequences, where those sequences hybridize specifically to sequences of complementary unnatural or orthogonal bases. Examples of unnatural bases and orthogonal base pairs are described herein, as well as in, for example, Hirao, I, et al. (A Synthetic Biology Approach to the Expansion of the Genetic Alphabet: Molecular Design of Unnatural Base Pairs of DNA TCIMAIL, No. 148, Tokyo Chemical Industry Co. Ltd., August 2012 and HIRAO, Ichiro, et al., Unnatural Base Pair Systems toward the Expansion of the Genetic Alphabet in the Central Dogma. Ed. Takao SEKIYA. Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 88.7 (2012): 345-367). Non-limiting examples of unnatural base pairs include: Isoguanine (isoG, 6-amino-2-ketopurine) with isocytosine (isoC, 2-amino-4-ketopyrimidine); xanthine with diaminopyridine; 2-amino-6-dimethylaminopurine (x) with 2-oxopyridine (y); 6-amino-5-nitro-3-(1-13-D-2-deoxyribofuranosyl)-2(1H)-pyridone (dZ) with 2-amino-8-(1--D-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP); 5SCIS with MMO2; 5SCIS with NaM (Malyshev, Denis A. et al. Solution Structure, Mechanism of Replication, and Optimization of an Unnatural Base Pair. Chemistry (Weinheim an Der Bergstrasse, Germany) 16.42 (2010): 12650-12659); with 7-(2-thienyl)imidazo[4,5-b]-pyridine (Ds) with pyrrole-2-carbaldehyde (Pa).
(47) RPNA and LPNA relate to other classes of nucleic acids, both natural and synthetic, in that it contains nucleobases (A, C, G, T, U, unnatural nucleobases, or other synthetic analogues) and thus is capable of hybridizing to its partner strand through hydrogen-bonding and base-stacking interactions, either in accordance with the Watson-Crick base-pairing rulein which the adenine nucleobase (A) is paired with thymine (T) or uridine (U), and cytosine (C) with guanine (G), or the equivalent Watson-Crick-like base-pairing for unnatural bases.
(48) LPNA differs from other classes of nucleic acids in that it adopts a left-handed helical motif and does not hybridize to complementary DNA or RNA strand, or any other classes of nucleic acids for that matter that fold into a right-handed helix, including all previously reported PNAs. LPNA will hybridize to another LPNA strand with a complementary sequence; however, it will not hybridize to DNA or RNA, or PNA prepared from L-amino acids (with the exception of L-serine, L-cysteine and related chiral amino acids, which can be made to fold into either a right-handed or left-handed helix depending on the nature of chemical transformations-another aspect of this invention), because of the conformational mismatch.
(49) One aspect of the present invention relates to the synthesis of gamma-peptide nucleic acids (PNAs) with a left-handed helical fold (LPNA). Unlike other classes of nucleic acid mimics that have been developed to date, which either adopt a random fold, such as glycol nucleic acid (GNA) and peptide nucleic acid (PNA), or a right-handed helical motif, such as phosphorothioate DNA, 2-O-methyl RNA, morpholino phosphoroamidate, locked nucleic acid (LNA), and all previously reported PNAs which were prepared from L-amino acids, this new class of chiral PNAs (or LPNA) adopt a left-handed helical motif and hybridize to one another with exquisite avidity and sequence specificity, in accordance with the Watson-Crick base-pairing rules.
(50) The left-handed helical fold renders LPNA impervious to hybridization with natural nucleic acid biopolymers such as DNA or RNA. This conformational orthogonality, combined with the exquisite binding affinity and sequence specificity and ease of synthesis and chemical group functionalization, makes LPNAs attractive as molecular tags for drug discovery (N. Winssinger, J. L. Harris, B. J. Backes, P. G. Schultz, Angew. Chem.-Int. Edit. Engl. 40, 3152 (2001); N. Winssinger, S. Ficarro, P. G. Schultz, J. L. Harris, Proc. Nat!. Acad. Sci. USA 99, 11139 (2002); F. Debaene, L. Mejias, J. L. Harris, N. Winssinger, Tetrahedron 60, 8677 (2004); J. L. Harris et al., Chern. Bioi. 11, 1361 (2004); and N. Winssinger et al., Chern. Bioi. 11, 1351 (2004)); recognition codes for organizing molecular self-assembly (J. H. Chen, N.C. Seeman, Nature 350, 631 (1991); S. M. Douglas et al., Nature 459, 414 (2009); Y.-W. Kwon, C. H. Lee, D.-H. Choi, J.-1. Jin, J Mater. Chern. 19, 1353 (2009); D. Y. Zhang, G. Seelig, Nat. Chern. 3, 103 (2011); F. A. Aldaye, A. L. Palmer, H. F. Sleiman, Science 321, 1795 (2008); and A. V. Pinheiro, D. Han, W. M. Shih, H. Yan, Nat. Biotech. 6, 763 (2011)); template-directed synthesis (Z. J. Gartner et al., Science 305, 1601 (2004); M. W. Kanan, M. M. Rozenman, K. Sakurai, M. M. Snyder, D. R. Liu, Nature 431, 545 (2004)); hybridization chain reaction (HCR) (R. M. Dirks, N. A. Pierce, Proc. Nat. Acad. Sci. US.A. 101, 15275 (2004); H. M. T. Choi et al., Nat. Biotech. 28, 1208 (2010)); logic gate/circuit designs (D. Y. Zhang, A. J. Turberfield, B. Yurke, E. Winfree, Science 318, 1121 (2007); G. Seelig, D. Soloveichik, D. Y. Zhang, E. Winfree, Science 314, 1585 (2006); B. M. Frezza, S. L. Cockroft, M. R. Ghadiri, JAm. Chern. Soc. 129, 14875 (2007); J. M. Picuri, B. M. Frezza, M. R. Ghadiri, JAm. Chern. Soc. 131, 9368 (2009); and 20. L. Qian, E. Winfree, Science 332, 1196 (2011)); molecular computations (S. M. Douglas, I. Bache let, G. M. Church, Science 335, 831 (2012); J. H. Reif, Science 332, 1156 (2011)); construction of nano-size materials and molecular devices (B. Yurke, A. J. Turberfield, A. P. Mills, F. C. Simmel, J. L. Neumann, Nature 406, 605 (2000); H. Z. Gu, J. Chao, S. J. Xiao, N. C. Seeman, Nature 465, 202 (2011); K. Lund et al., Nature 465, 206 (2010)); development of biomaterials for tissue engineering and regenerative medicine (L. G. Griffin, G. Naughton, Science 295, 1009 (2002)); capturing reagents for biomolecule (DNA, RNA, proteins, carbohydrates, and other biological machineries); pull-down and purification (B. van Steensel, J. Dekker, Nat. Biotech. 28, 1089 (2010); W. A. Whyte et al., Cell 153, 307 (2013)); and peptide therapeutics (S. Rapireddy et al., JAm. Chern. Soc. 134, 4041 (2012); A. G. Poth et al., ACS Chern. Bioi. 6, 345 (2011); S. H. Gellman, Ace. Chern. Res. 31, 173 (1998); D. J. Hill, M. J. Mio, R. B. Prince, T. S. Hughes, J. S. Moore, Chern. Rev. 101, 3893 (2001)).
(51) Because LPNA does not hybridize to a complementary DNA or RNA strand, it can be used in many of the aforementioned applications where hybridization of probes or molecular recognition tags to the endogenous nucleic acid molecules such as DNA or RNA is a concern. One such example is the construction of molecular circuits (or logic gates) in live cells and intact organisms for detection and treatment of genetic and infectious diseases. Of particular significance is the application of PNA to convert a right-handed input into a left-handed PNA output, and vice versa, via a chain-migration mechanism. A second example is hybridization chain reaction (an in-situ signal amplification method) for detection of nucleic acids (such as DNA and RNA) and other biopolymers. A third example is combinatorial synthesis and screening of chemical libraries in drug discovery. In all cases, unintended hybridization of probes or molecular recognition tags to endogenous nucleic acid molecules such as DNA and RNA, which is a likely scenario due to their high abundance in the cellular environments, would lead to non-productive binding events, or disruption in the signals (or messages) being relayed or the structures being built, due to inadvertent sequestration of the probes or molecular recognition tags.
(52) Because of its conformational and hybridization orthogonality, LPNA can be used to program molecular recognition and self-assembly in the cellular environments without the concern for inadvertent binding of probes with endogenous nucleic acid materials. Also, because of its extraordinary high binding affinity, relatively short probes (3-8 nucleotides in length) can be used to perform many of the aforementioned molecular recognition and self-assembly tasks. This provides many distinct advantages over the longer ones (typically 15-50 nucleotides in length commonly used today) in terms of ease of synthesis, cost of production, recognition specificity, and cell penetration and systemic delivery. Other advantages that LPNA (as well as RPNA) has over the other classes of nucleic acid mimics are ease and flexibility of chemical modifications and enzymatic stability. The ability to incorporate other chemical functionalities into LPNA with ease is essential to further fine-tuning and improving its properties toward a particular application in biology and medicinea task that would otherwise be difficult (if not impractical) to achieve with other classes of nucleic acid molecules.
(53) LPNA can be chemically derivatized in such a way that it is soluble in organic solvents, such as dichloromethane, acetonitrile, tetrahydrofuran (THF), and hexane. Other nucleic acid molecules, even those as small as individual building blocks, without the nucleobase protection (which is required for base-pairing) are generally not miscible with these commonly used organic solvents. Because LPNA can be soluble in organic solvents, it can be used to organize and assemble smart polymeric and macro-scale materials whose structures and functions are determined by the input stimuli because Watson-Crick recognition is still maintained in these solvents.
(54) Further, since LPNA is synthetic, made of unnatural polyamide backbone, it is not recognized or easily degraded by proteases or nucleases. As such, it can be used in many of these intracellular (or in vivo) applications without the concern for enzymatic degradation that would render the probes useless, or in the case of randomly-folded and right-handed helical nucleic acids, inadvertent binding to endogenous nucleic acid materials that would cause hybridization short-circuit or elicit undesired molecular consequences.
(55) With regard to synthesis of PNAs, the synthetic route, as shown in Example 2, below, enables the production of both the right-handed (RPNA) as well as the left-handed (LPNA) monomers on a commercial scale from a common and relatively cheap starting material (L-serine in this case, but also applicable to L-cys), without the concern for racemization.
(56) There are currently other methods for detection of nucleic acids. Current methods for nucleic acid-based diagnostics and the respective advantages and disadvantages of each are described below. Further, Tables 1 and 2, below, provide a partial list of Human Genetic Tests and Microbial Tests, respectively, based on nucleic acid detections currently on the U.S. market. However, unlike the methods described herein, these methods are not performed in-situ. Further, the current methods are labor intensive, time-consuming, prone to cross-contamination and false positives, and also require the use of an enzyme. As such, there exists a need for a rapid, convenient means for diagnosing genetic and infectious diseases in a laboratory setting as well as in the field.
(57) Nucleic acid-based diagnostics generally rely on four core assay technologies. In PCR or RT-PCR, DNA (or RNA reverse transcribed to cDNA) is amplified to detect the presence of a pathogen or host gene of interest. This allows for sensitive detection, but requires a highly skilled technician and laboratory equipment. The amplified material can contaminate subsequent samples. Isothermal amplification is similar to PCR, but uses simplified amplification techniques that operate at a single temperature. Examples of isothermal methods include: Transcription mediated amplification (TMA); Signal-mediated amplification of RNA technology (SMART); Strand displacement amplification (SDA); Rolling circle amplification (RCA); Loop-mediated isothermal amplification of DNA (LAMP); Isothermal multiple displacement amplification (HAD); Single primer isothermal amplification (SPIA); Circular helicase-dependent amplification (cHDA); and Branched DNA signal amplification (bDNA). In these assays, DNA is amplified which allows for sensitive detection, simplified isothermal amplification is more amenable to point-of-care devices/low-resource settings, however, this newer technology that has not been extensively clinically validated, amplified material can contaminate subsequent samples. Hybridization techniques, such as FISH and DNA microarray technologies benefit from no risk of contamination of new samples with amplified material, but because the target DNA is not amplified, the techniques can be less sensitive and requires highly skilled technician and laboratory equipment. Sequencing involves identification of the specific genetic code (sequence) for an amplified piece of DNA or the full genome of an organism. Beyond confirming the presence of a pathogen, sequencing can provide detailed information about the original and unique characteristics of the pathogen in a specific patient, not practical for low-resource settings. However, sequencing techniques are expensive, time consuming, and require both a highly skilled technician and expensive laboratory equipment.
(58) A multitude of nucleic acid-based assays are known and are commercially-available, and the target nucleic acids and sequences thereof are broadly-known. Those unique sequence can be utilized as target sequences for the assays described herein, such as the I-NADSA assay described below. A large number of commercially-available nucleic acid-based assays are listed in Tables 2 and 3 of parent patent application, U.S. Provisional Patent Application No. 61/996,483, which is incorporated herein by reference in its entirety (see, Tables 1 and 2, below, providing a partial listing of those assays provided in Tables 2 and 3 of U.S. Provisional Patent Application No. 61/996,483, which is publicly available from the U.S. Food and Drug Administration as a list of nucleic acid-based tests that have been cleared or approved by the Center for Devices and Radiological Health). Listing the many thousands of developed and/or commercially available PCR, isothermal amplification, hybridization and target-specific probes herein is unnecessary, as one of ordinary skill can either use the same unique sequences used in the assay as the target sequence for the assays described herein, or can readily ascertain if any sequence of any target gene is unique in a genome, transcriptome or exome by either searching a publicly-available database, such as GenBank, for the target sequence, or by conducting a preliminary hybridization or PCR experiment using the target sequence to ascertain if there will be cross-reactivity with other sequences in a sample to be analyzed.
(59) TABLE-US-00001 TABLE 1 Human Genetic Tests (Nucleic Acid-Based Detections) Manufacturer Disease Trade name Method Submission Abbot Molecular, Inc. Acute myeloid Vysis D7S486/CEP 7 FISH FISH K131508 leukemia Probe Kit Cytogenetic Vysis EGR1 FISH Probe Kit FISH K123951, Cytogenetic K091960 Nanostring Technologies Breast cancer Prosigna Breast Cancer Hybridization K130010 Prognostic Gene Signature Assay Gene profiling Illumina, Inc. Cystic fibrosis Illumina MiSeqDx Fibrosis Sequencing K132750 Clinical Sequencing Assay Illumina MiSeqDx Cystic Sequencing K124006 Fibrosis 139-Variant Assay Luminex Molecular Drug metabolizing xTAG CYP2D6 Kit v3 PCR & K1301189, Diagnostics, Inc. enzymes Hybridization K093420 Hologics, Inc. Coagulation Invading Factor V Hybridization K100980 factors SNP Invading Factor II Hybridization K100943 SNP Invader MTHFR 677 Hybridization K100987 SNP Invader MTHFR 1298 Hybridization K100496 SNP xDx Heart transplant AlloMap Molecular RT-PCR K073482 Expression Testing Gene profiling Affymetrix, Inc. Chromosome Affymetrix CytoScan Dx Assay Hybridization K130313 abnormalities Gene profiling Iris Molecular Diagnostics Prostate cancer NADiA ProsVue qPCR K101185
(60) TABLE-US-00002 TABLE 2 Microbial Tests (Nucleic Acid-Based Detections) Manufacturer Microorganisms Trade name Method Submission Argene SA Adenovirus Adenovirus R-gene US RT-PCR K121942 Idaho Bacillus Anthracis IBAIDS Anthrax RT-PCR K131930, Technology, Inc. Detection System K071188, K051713 Microprobe Corp. Candida Affirm VPIII Microbial K931151, tropicalis/Candida Identification Test K931374 albicans/Candida parapsilosis/Candida glabrata/Candida krusei PrimeraDx Clostridium difficile ICEPlex C. difficile Kit PCR, separation K132726 Idaho Coxiella burnetii Joint Biological Agent RT-PCR K103207 Technology, Inc. Identification and Diagnostics System (JBAIDS) Q Fever Detection Kit Hologic/ Chlamydia Aptima Combo 2 Assay NA Amplification K132251 Gen-Probe, Inc. trachomatis/Neisseria gonorrhoeae Roche Molecular Cytomegalovirus COBAS RT-PCR P110037, Systems AmpliPrep/COBAS S001-S002 TaqMan CMV Test Center for Disease Dengue virus CDC DENV-1-4 Real-Time TaqMan K113336 Control and RT-PCR Assay Prevention Intelligent Medical Entercoccus IMDx Van R for Abbott PCR K123753 Devices, Inc. m2000 bioMerieux, Inc. Enterovirus NucliSens EasyQ RT-PCR K093383 Enterovirus vl.1 Assay AdvanDx Escherichia GNR Traffic Light K101558 coli/Klebsiella PNA FISH pneumonia/Pseudomonas E. coli/P. aeroginosa K081309, aeroginosa PNA FISH K092393 EK/P aeruginosa PNA FISH K092393, K081433 E. coli PNA FISH K082068 Idaho Technology, Francisella tularensis JBAIDS Tularemia K072547 Inc. Detection Kit Nanosphere, Inc. Gram-Positive/Gram- Verigene Gram-Positive K122514, Negative Bacteria Herpes Blood Culture Nucleic K113450 Simplex Virus Acid Test (BC-GP) Abbott Molecular, Hepatitis virus Abbot RealTime HCV P120012 Inc. Genotype II Abbott RealTime HCV P100017, Assay S001-S006 Abbott RealTime HBV P080026, Assay S001-S004 Quidel Corporation Human Quidel Molecular RSV + K131813, Metapneumovirus hMPV Assay K122189 Quidel Molecular K112490 hMPV Assay Gen-Probe, Inc. Human papillomavirus Aptima HPV Assay P100042 Aptima HPV 16 18/45 P120007 Genptype Assay Gen-Probe Prodesse, Inc. Influenza and respiratory Prodesse ProFAST Assay K132237 viruses US Army Medical Leishmania species SMART Leish K081868 Material Development Activity Cepheid Mycobacterium tuberculosis Xpert MTB/RIF Assay M131706 Gen-Probe, Inc. Mycobacterium species Accuprobe Mycobacterium K921435, avium complex culture K896494, K897078 Accuprobe Mycobacterium K904463 kansasii Identification Test Accuprobe Mycobacterium K897077 intracellular Culture Identification Test Accuprobe Mycobacterium K896492 gordonae culture identification Test Rapid Diagnostic System for K890089 Mycobacterium gordonae Rapid Diagnostic System K864597 for Mycobacteria Rapid Identification Test K862613 for Mycobacterium avium Gen-Probe Mycobacterium K860782 Rapid Confirmation System Meridian Mycoplasma Illumigene Mycoplasma K123423 Bioscience, Inc. pneumonia DNA Amplification Assay Multiplex Panel BD Diagnostics Staphylococcus BD Max MRSA Assay K120138 (GeneOhm Sciences Canada, Inc.) Cepheid Streptococci Xpert GBS LB Assay K121539 Gen-Probe, Inc. Trichomonas vaginalis Aptima Trichomonas K102911 vaginalis assay Idaho Technology, Yersinia pestis JBAIDS Plague Detection K072631 Inc. Kit HPA: Hybridization protection assay LCR: Ligase chain reaction
(61) An example of where the paradigm early detection equals better outcomes is especially true is the syndrome sepsis, a continuum of events triggered by the body's inflammatory immune responses to bacterial, viral, fungal, or parasitic infections. Sepsis is a major killer globally, despite significant advances in treatment of infectious disease and improvements in clinical care. Once a patient is septic, the survival rate drops 6% every hour. Therefore, early diagnosis of sepsis is important for patient survival. Various analytes show promise as markers for identifying septic patients. Tallia S, Kunicka J E. Sepsis: Improving the Odds. Perspectives; Spring 2009: 6-11. However, there is still a need for faster and more reliable tests and markers for a quick diagnosis, e.g. less than 30 minutes from testing.
(62) As used herein, the terms drug and drugs refer to any compositions having a preventative or therapeutic effect, including and without limitation, antibiotics, peptides, hormones, organic molecules, vitamins, supplements, factors, proteins and chemoattractants.
(63) As used herein, the terms cell and cells refer to any types of cells from any animal, such as, without limitation, rat, mice, monkey, and human. For example and without limitation, cells can be progenitor cells, such as stem cells, or differentiated cells, such as endothelial cells, smooth muscle cells. In certain embodiments, cells for medical procedures can be obtained from the patient for autologous procedures or from other donors for allogeneic procedures.
(64) By expression or gene expression, it is meant the overall flow of information from a gene (without limitation, a functional genetic unit for producing a gene product, such as RNA or a protein in a cell, or other expression system encoded on a nucleic acid and comprising: a transcriptional promoter and other cis-acting elements, such as response elements and/or enhancers; an expressed sequence that typically encodes a protein (open-reading frame or ORF) or functional/structural RNA, and a polyadenylation sequence), to produce a gene product (typically a protein, optionally post-translationally modified or a functional/structural RNA). By expression of genes under transcriptional control of, or alternately subject to control by, a designated sequence, it is meant gene expression from a gene containing the designated sequence operably linked (functionally attached, typically in cis) to the gene. The designated sequence may be all or part of the transcriptional elements (without limitation, promoters, enhancers and response elements), and may wholly or partially regulate and/or affect transcription of a gene. A gene for expression of a stated gene product is a gene capable of expressing that stated gene product when placed in a suitable environmentthat is, for example, when transformed, transfected, transduced, etc. into a cell, and subjected to suitable conditions for expression. In the case of a constitutive promoter suitable conditions means that the gene typically need only be introduced into a host cell. In the case of an inducible promoter, suitable conditions means when an amount of the respective inducer is administered to the expression system (e.g., cell) effective to cause expression of the gene.
(65) Provided herein are sequence-based logic reactions that allow for extremely accurate molecular events and detection within a complex system such as within a cell, cell lysate, tissue, blood, saliva, bodily fluid (such as sweat), urine, or feces. U.S. Pat. No. 8,630,809 describes a simple type of such sequence-based logic reactions. In the methods and compositions described herein, by incorporating a conversion from native R configured helical structures and conformations, to L configured structures, the system is simplified, cleverly sidestepping the possibility of competing reactions and reactants that may be present in a system, such as in the system described in U.S. Pat. No. 8,630,809.
(66) The system described herein relies in one aspect on the ability of nucleic acids to hybridize both to achiral and RPNA, but not to LPNA. As above,
(67)
(68) According to one aspect of the methods described herein, using sequence-specific binding reactions, presence of a nucleic acid comprising a specific nucleobase sequence is converted via achiral PNA to an LPNA initiator, which triggers an amplification cascade using two or more LPNA hairpin amplifiers that concatenate only in the presence of the LPNA initiator. The concatenation event is detected by any useful means, such as, without limitation, by gel, UV spectroscopy, light scattering spectroscopy (e.g., dynamic light scattering (DLS)), rheological methods (viscosity), colorimetric assay, FRET analysis, fluorescence dequenching methods (e.g., molecular beacon), fluorescence activation, enzyme-linked immunosorbent assay (ELISA), or tyramide signal amplification (TSA). FRET analysis and excimer formation are described below in the Examples.
(69) According to one aspect of the disclosure, referencing
(70) The in situ method involves adding the reagents to a cell or tissue sample. The method of identifying a target nucleic acid in a sample comprising nucleic acids involves mixing the sample with the reagents. The method of amplifying a target nucleic acid sequence involves contacting a target nucleic acid with the reagents. All three methods include a detection step for detecting the production of the displaced LPNA, as disclosed herein, for example by a hybridization chain reaction. Nucleic acid, biological sample and in situ cell and tissue preparation methods are extremely well-known and need not be disclosed herein to fully understand the disclosed invention.
(71) In another aspect of the disclosure a composition is provided for use in detecting a nucleic acid. The composition comprises, referencing
(72) According to yet another aspect, the composition is provided in a kit, which comprises the composition in a vessel, such as an Eppendorf tube, multi-well plate, array, etc., in packaging, such as a box, foil pack, plastic bag, envelope, etc. acceptable for shipping. The vessel may form part of or consist of a cartridge, such as a disposable for use in an automated system for conducting assays, involving suitable robotics, fluidics, optics, etc., as are broadly available commercially for processing biological samples, for example by mixing liquids and measuring a reaction, e.g., by fluorescence detection.
(73) According to one aspect of the invention, an analyte is detected by a hybridization chain reaction. A hybridization chain reaction, in a simpler form from the reaction described herein, is described for example and without limitation in U.S. Patent Publication No. 2005/02606351 A1, describing use of metastable nucleic acid hairpin monomers that are used in pairs or in combination of two or more hairpin structures. Although the structure of the metastable nucleic acid hairpin monomers is described in the context of nucleic acids and to some limited extent peptide nucleic acids, the overall sequence structure and logic of the formation of a detection of concatamerized products of the described monomers is equally applicable to the LPNA amplifiers described herein, which are analogous to the monomers of that publication. The monomers described in U.S. 2005/0260635 are described as being metastable because in the absence of the initiator, they are kinetically disfavored from associating with other monomer(s), but in the presence of the initiator, the monomers form a nicked double helix. Likewise, the LPNA amplifiers described herein do not concatamerize in the absence of the initiatorthe LPNA from the converter.
(74) Identification of the concatamerization of the described LPNA hairpin amplifiers by hybridization chain reaction, or identification of the presence of a target sequence in a nucleic acid by any method described herein can be accomplished by a variety of methods. The products can be identified, for example by gel electrophoresis, spectrometer, rheological methods, colorimetric methods, FRET methods, fluorescent dequenching methods (e.g., molecular beacon), fluorescent activation, enzyme-linked immunosorbent assay (ELISA), tyramide signal amplification (TSA), etc. See, generally, Marras, SAE, Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes, Methods in Molecular Biology 335:3-16 (2006). As shown in the Example below, a FRET pair may be used, with the donor being located on the first amplifier and the acceptor being located on the second amplifier, such that only when the first and second amplifiers concatamerize, the acceptor will fluoresce when the donor is excited. Thus, as is broadly known in the arts, the donor has an excitation spectrum in a shorter wavelength than the excitation spectrum of the acceptor (they overlap insignificantly, and preferably not at all), the donor has an emission spectrum that significantly overlaps (typically >50%) with the excitation spectrum of the acceptor, and the acceptor emission spectrum is discernable from the emission spectrum, and preferably does not overlap. The donor typically has a high extinction coefficient and a high quantum yield. FRET typically works when the effective distance between donor and acceptor is between 10 and 100 . As such one of ordinary skill can select adequate FRET pairs from the multitude of chromophores available commercially. A non-limiting list of examples of common FRET donor/acceptor pairs include: fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate; Cy3/Cy5; Enhanced Green Fluorescent Protein (EGFP)/Cy3; cyan fluorescent protein/yellow fluorescent protein (YFP); and EGFP/YFP.
(75) In another aspect, excimer formation may be used to detect concatamerization, as in the assay described in the Example below. An excimer is a composition that either does not fluoresce or that fluoresces at a different wavelength when in proximity with another molecule, which may be the same molecule. In the Example below, the emission spectrum of pyrene monomer is shown to shift100 nm when dimerized.
(76) Another method by which concatamerization may be detected is by fluorescent dequenching. In one example, molecular beacon technology can be utilized, as is broadly-known in the arts. In this example, the amplifiers comprise both a fluorophore and a quencher of the fluorophore that only quenches the fluorophore when the amplifier is in its hairpin configuration. The fluorophore and quencher are located on the amplifier such that when the hairpin is opened, and the amplifiers are concatamerized, the quencher no longer quenches the fluorescence of the fluorophore. In an alternate embodiment, the quencher is located on one amplifier and the fluorophore is located on the other, and are positioned such that when not concatamerized, the fluorophore fluoresces, and when concatamerized, the fluorophore is quenched. Another version of fluorescent dequenching is described in, for example, Pianowski, Z, et al., Imaging of mRNA in Live Cells Using Nucleic Acid-Templated Reduction of Azidorhodamine Probes, J. Am. Chem. Soc. 131:6492-6497 (2009), in which a profluorescent, chemically modified fluorophore, is modified by an activator on the other strand to produce a fluorescent moiety. In the specific example provided in that article, profluorescent azidorhodamine is attached to a first strand, and is unmasked by hybridization to a nucleic acid strand having a pendant alkylphosphine. As described in Pianowski, Z, et al., azidocoumarin dyes also may be unmasked by an alkyl phosphine.
(77) In a further aspect of detection of the concatamerization, fluorescence activation may be used, in which a portion of a fluorophore is located on each strand, with each portion being non-fluorescent or less fluorescent at a detection excitation and/or emission wavelength, and which, when placed in proximity, the portions covalently link and form a fluorochrome that fluoresces at the detection excitation and emission wavelengths. A non-limiting example of this is the Cu(I)-mediated click-chemistry linking of azide and alkyne moities described in Sivakumar, K, et al., A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-Azidocoumarins and Acetylenes, Organic Letters, 6(24):4603-4606 (2004), in which 3-azido-coumarin and acetylene- (ethyne-) substituted phenyl or substituted phenyl profluorophores are linked in the presence of Cu(I) (e.g., CuSO.sub.4) to form a fluorphore. In the context of the compositions described herein, and for example in the I-NADSA reaction described below, the 3-azidocoumarin profluorophore is linked to one amplifier, and the acetylene-substituted phenyl profluorophore is linked to the other amplifier, and are located such that when Cu(I) is added to the reaction, typically after addition of a nucleic acid sample to be analyzed (e.g., 30 seconds, 1, 2, 3, 4, 5, 10, or 15 minutes or increments therebetween), profluorophore combinations (that is, the 3-azidocoumarin and the acetylene-substituted phenyl moieties) present in the concatamerized product, will link covalently to form a triazolylcoumarin fluorophore. Examples of suitable azide and alkyne building blocks (profluorophores) useful in any combination and which are useful for detection of concatamerization of the amplifiers in the described I-NADSA reaction described herein, are provided in
(78) ##STR00006##
where R5 is H, (C.sub.1-C.sub.6) alkyl, phenyl, (C.sub.1-C.sub.6) alkoxyl, halo-substituted (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkyl ester, e.g., Cl, I, F, or Br, and R6 is H, halo, (C.sub.1-C.sub.6) alkyl, (C.sub.1-C.sub.6) alkoxyl, halo-substituted (C.sub.1-C.sub.6) alkyl, halo-substituted (C.sub.1-C.sub.6) alkyl, or amine.
(79) In another aspect, as indicated in
(80) As used herein, an amino acid side chain is a side chain for an amino acid. Amino acids have the structure:
(81) ##STR00007##
where R is the amino acid side chain. Non-limiting examples of amino acid side chains are shown in
(82) Alkyl refers to straight, branched chain, or cyclic hydrocarbon groups including from 1 to about 20 carbon atoms, for example and without limitation C.sub.1-3, C.sub.1-6, C.sub.1-10 groups, for example and without limitation, straight, branched chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like. Substituted alkyl refers to alkyl substituted at 1 or more, e.g., 1, 2, 3, 4, 5, or even 6 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein. Optionally substituted alkyl refers to alkyl or substituted alkyl. Halogen, halide, and halo refers to F, CI, Br, and/or I. Alkylene and substituted alkylene refer to divalent alkyl and divalent substituted alkyl, respectively, including, without limitation, ethylene (CH.sub.2CH.sub.2). Optionally substituted alkylene refers to alkylene or substituted alkylene.
(83) Alkene or alkenyl refers to straight, branched chain, or cyclic hydrocarbyl groups including from 2 to about 20 carbon atoms, such as, without limitation C.sub.1-3, C.sub.1-6, C.sub.1-10 groups having one or more, e.g., 1, 2, 3, 4, or 5, carbon-to-carbon double bonds. Substituted alkene refers to alkene substituted at 1 or more, e.g., 1, 2, 3, 4, or 5 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein. Optionally substituted alkene refers to alkene or substituted alkene. Likewise, alkenylene refers to divalent alkene. Examples of alkenylene include without limitation, ethenylene (CHCH) and all stereoisomeric and conformational isomeric forms thereof. Substituted alkenylene refers to divalent substituted alkene. Optionally substituted alkenylene refers to alkenylene or substituted alkenylene.
(84) Alkyne or alkynyl refers to a straight or branched chain unsaturated hydrocarbon having the indicated number of carbon atoms and at least one triple bond. Examples of a (C.sub.2-C.sub.8)alkynyl group include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below. The term alkynylene refers to divalent alkyne. Examples of alkynylene include without limitation, ethynylene, propynylene. Substituted alkynylene refers to divalent substituted alkyne.
(85) The term alkoxy refers to an O-alkyl group having the indicated number of carbon atoms. For example, a (C.sub.1-C.sub.6)alkoxy group includes O-methyl (methoxy), O-ethyl (ethoxy), O-propyl (propoxy), O isopropyl (isopropoxy), O-butyl (butoxy), O-sec-butyl (sec-butoxy), O-tert-butyl (tert-butoxy), O-pentyl (pentoxy), O-isopentyl (isopentoxy), O-neopentyl (neopentoxy), O-hexyl (hexyloxy), O-isohexyl (isohexyloxy), and O-neohexyl (neohexyloxy). Hydroxyalkyl refers to a (C.sub.1-C.sub.10)alkyl group wherein one or more of the alkyl group's hydrogen atoms is replaced with an OH group. Examples of hydroxyalkyl groups include, but are not limited to, CH.sub.2OH, CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2OH, and branched versions thereof.
(86) The term ether or oxygen ether refers to an alkyl group, such as a (C.sub.1-C.sub.10)alkyl group wherein one or more of the alkyl group's carbon atoms is replaced with an O group. An example of an ether is a poly(alkylene oxide), also referred to as a poly(alkylene glycol) or polyglycols, such as a poly(C.sub.1-C.sub.10) alkylene oxide), such as poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(butylene glycol) (PBG), etc., and include two or more monomer residues, for example in one aspect, from 2-50 monomer residues. The term ether includes CH.sub.2(OCH.sub.2CH.sub.2).sub.qOP.sub.1 compounds where P1 is a protecting group, H, or a (C.sub.1-C.sub.10)alkyl. Exemplary ethers include polyethylene glycol, diethylether, methylhexyl ether, PEG-PPG-PEG or PPG-PEG-PPG block copolymers, etc.
(87) The term thioether refers to (C.sub.1-C.sub.10)alkyl group wherein one or more of the alkyl group's carbon atoms is replaced with an S group. The term thioether includes CH.sub.2(SCH.sub.2CH.sub.2).sub.qSP.sub.1 compounds where P1 is a protecting group, H, or a (C.sub.1-C.sub.10)alkyl. Exemplary thioethers include dimethylthioether, ethylmethyl thioether. Protecting groups are known in the art and include, without limitation: 9-fluorenylmethyloxy carbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzhydryloxycarbonyl (Bhoc), benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn), allyloxycarbonyl (alloc), trityl (Trt), dimethoxytrityl (DMT), 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde), diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts) and monomethoxytrityl (MMT) groups.
(88) Aryl, alone or in combination refers to an aromatic monocyclic or bicyclic ring system such as phenyl or naphthyl. Aryl also includes aromatic ring systems that are optionally fused with a cycloalkyl ring. A substituted aryl is an aryl that is independently substituted with one or more substituents attached at any available atom to produce a stable compound, wherein the substituents are as described herein. Optionally substituted aryl refers to aryl or substituted aryl. Arylene denotes divalent aryl, and substituted arylene refers to divalent substituted aryl. Optionally substituted arylene refers to arylene or substituted arylene.
(89) Heteroatom refers to N, O, P and S. Compounds that contain N or S atoms can be optionally oxidized to the corresponding N-oxide, sulfoxide or sulfone compounds. Hetero-substituted refers to an organic compound in any embodiment described herein in which one or more carbon atoms are substituted with N, O, P or S.
(90) Cycloalkyl refer to monocyclic, bicyclic, tricyclic, or polycyclic, 3- to 14-membered ring systems, which are either saturated, unsaturated or aromatic. The cycloalkyl group may be attached via any atom. Cycloalkyl also contemplates fused rings wherein the cycloalkyl is fused to an aryl or hetroaryl ring. Representative examples of cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group can be unsubstituted or optionally substituted with one or more substituents as described herein below. Cycloalkylene refers to divalent cycloalkyl. The term optionally substituted cycloalkylene refers to cycloalkylene that is substituted with 1, 2 or 3 substituents, attached at any available atom to produce a stable compound, wherein the substituents are as described herein.
(91) Carboxyl or carboxylic refers to group having the indicated number of carbon atoms and terminating in a C(O)OH group, thus having the structure RC(O)OH, where R is a divalent organic group that includes linear, branched, or cyclic hydrocarbons. Non-limiting examples of these include: C.sub.1-8 carboxylic groups, such as ethanoic, propanoic, 2-methylpropanoic, butanoic, 2,2-dimethylpropanoic, pentanoic, etc.
(92) (C.sub.3-C.sub.8)aryl-(C.sub.1-C.sub.6)alkylene refers to a divalent alkylene wherein one or more hydrogen atoms in the C.sub.1-C.sub.6 alkylene group is replaced by a (C.sub.3-C.sub.8)aryl group. Examples of (C.sub.3-C.sub.8)aryl-(C.sub.1-C.sub.6)alkylene groups include without limitation 1-phenylbutylene, phenyl-2-butylene, l-phenyl-2-methylpropylene, phenylmethylene, phenylpropylene, and naphthylethylene. The term (C.sub.3-C.sub.8)cycloalkyl-(C.sub.1-C.sub.6)alkylene refers to a divalent alkylene wherein one or more hydrogen atoms in the C.sub.1-C.sub.6 alkylene group is replaced by a (C.sub.3-C.sub.8)cycloalkyl group. Examples of (C.sub.3-C.sub.8)cycloalkyl-(C.sub.1-C.sub.6)alkylene groups include without limitation 1-cycloproylbutylene, cycloproyl-2-butylene, cyclopentyl-1-phenyl-2-methylpropylene, cyclobutylmethylene and cyclohexylpropylene.
(93) A polymer is a homopolymer or copolymer prepared from monomers, which, when incorporated into the polymer are residues. Polymers may have a variety of topologies, such as linear, branched, circular, comb, star, etc. Polymers include block copolymers.
(94) Unless otherwise indicated, the nucleic acids and nucleic acid analogs described herein are not described with respect to any particular sequence of bases. The present disclosure is directed to generally and broadly-applicable methods and compositions, and the usefulness of any specific embodiments described herein, while depending upon a specific sequence in each instance, is generically and broadly applicable to any particular sequence of nucleobases and sequences complementary thereto. Based on the abundance of published work with nucleic acids, nucleic acid analogs and PNA (e.g., PNA), it is expected that any nucleobase sequence attached to the backbone of the described PNA oligomers would hybridize in an expected, specific manner with a complementary nucleobase sequence of a target nucleic acid or nucleic acid analog by Watson-Crick or Watson-Crick-like hydrogen bonding. One of ordinary skill would understand that the compositions and methods described herein are sequence-independent.
(95) The nucleic acids, nucleic acid analogs, and PNAs described herein comprise a backbone and at least two nucleobases. This structure is shown schematically in Formula 3:
(96) ##STR00008##
where R1 is a backbone monomer residue and R2s are, independently nucleobases. E are independently end (terminal) groups that are part of the terminal monomer residues, and n is any positive integer or 0. Typically, all instances of R1 are the same with the exception of the terminal monomer residues which typically have different end-groups E as compared to internal monomers, such as, without limitation NH.sub.2 and C(O)OH or CONH.sub.2 at the respective N-terminal and C-terminal ends for PNAs, and hydroxyl groups at the 5 and 3 ends of nucleic acids. An exception to this is the P-strand described herein as having a terminal sulthydryl or protected sulthydryl, and an internal thioester bond.
(97) In one aspect of the present invention, reagents, such as the nucleic acids, nucleic acid analogs, and/or PNAs are implemented as a solution-phase assay, that is, as in Example 4, all reagents are present in solution, with no reagent being bound to a surface, as in an array, below.
(98) In one aspect of the present invention, reagents, such as the nucleic acids, nucleic acid analogs, and/or PNAs are implemented on a substrate or an array. Arrays are particularly useful in implementing high-throughput assays, such as genetic detection assays. As used herein, the term array refers to reagents, for example the PNA reagents described herein, located at two or more discrete, identifiable and/or addressable locations on a substrate. In one embodiment, an array is an apparatus having two or more discrete, identifiable reaction chambers, such as, without limitation a 96-well dish, in which reactions comprising identified constituents are performed. In an exemplary embodiment, two or more reagents described herein are immobilized onto a substrate in a spatially addressable manner so that each individual set of PNAs, e.g., the sensor, converter and amplifiers are located at a different and identifiable (addressable) location on the substrate. Substrates include, without limitation, multi-well plates, silicon chips and beads. In one embodiment, the array comprises two or more sets of beads, with each bead set having an identifiable marker, such as a quantum dot or fluorescent tag, so that the beads are individually identifiable using, for example and without limitation, a flow cytometer. In one embodiment, an array is a multi-well plate containing two or more wells with the described reagents for amplifying specific sequences. As such, reagents, such as one or more of the sensor, converter and amplifiers may be bound or otherwise deposited onto or into specific locations on an array. Reagents may be in any suitable form, including, without limitation: in solution, dried, lyophilized or glassified. When linked covalently to a substrate, such as an agarose bead or silicon chip, a variety of linking technologies are known for attaching chemical moieties, such as the PNA compositions described herein, to such substrates. Linkers and spacers for use in linking nucleic acids, peptide nucleic acids and other nucleic acid analogs are broadly known in the chemical and array arts and for that reason are not described herein. As a non-limiting example, a PNA reagent contains a reactive amine, which can be reacted with carboxyl, cyanogen bromide-, N-hydroxysuccinimide ester-, carbonyldiimidazole- or aldehyde-functional agarose beads, available, for instance from Thermo Fisher Scientific (Pierce Protein Biology Products), Rockford, Ill. and a variety of other sources. The reagents described herein can be attached to a substrate in any manner, with or without linkers. In one aspect, the LPNA strand of the converter is linked to the substrate. Informatics and/or statistical software or other computer-implemented processes for analyzing array data and/or identifying genetic risk factors from data obtained from a patient sample, are known in the art. In a further, non-arrayed embodiment, reagents, e.g., PNAs for carrying out only one specific assay, are contained within a single vessel or well, with, e.g., the PNA of the converter strand, is linked to the substrate/vessel/tube/well.
(99) The reactions described herein can be efficiently multiplexed, so long as the sequences do not cross-react and the detection method can differentiate the products of the different reactions, e.g., by employing different fluorochromes, such that the excitation and/or emission wavelengths of the signal for detection of the concatenated product are different.
(100) By immobilized in reference to a composition such as a nucleic acid, nucleic acid analog, or PNA as described herein, it is meant attached to a substrate of any physical structure or chemical composition. The immobilized composition is immobilized by any method useful in the context of the end use. The composition is immobilized by covalent or non-covalent methods, such as by covalent linkage of amine groups to a linker or spacer, or by non-covalent bonding, including Van derWaals and/or hydrogen bonding. A label is a chemical moiety that is useful in detection of, or purification or a molecule or composition comprising the label. A label may be, for example and without limitation, a radioactive moiety, such as .sup.14C, .sup.32P, .sup.35S, a fluorescent dye, such as fluorescein isothiocyanate or a cyanine dye, an enzyme, or a ligand for binding other compounds such as biotin for binding streptavidin, or an epitope for binding an antibody. A multitude of such labels, and methods of use thereof are known to those of ordinary skill in the immunology and molecular biology arts.
(101) In another aspect of the present disclosure, a method of preparing optically pure LPNA and RPNA monomers are provided. The method is a distinct improvement over current methods, and substantially increases the commercial viability of use of chiral PNAs. As such a method for preparing a chiral PNA monomer is provided. Further to Example 2, the method comprises first preparing a dibenzylated intermediate by dibenzylating the amino group of L-serine; protecting the hydroxyl group of the side chain of the L-serine; and reducing the carboxylic acid group of the L-serine to obtain a dibenzylated intermediate having a protected hydroxyl group. The dibenzylated intermediate can then be used to prepare either left-handed or right-handed PNA monomers. To prepare right-handed PNA monomers, the method comprises: mesylating, and azidatizing the dibenzylated intermediate and deprotecting the protected hydroxyl group of the dibenzylated intermediate to obtain an azidated intermediate having an azido group and a hydroxyl group; alkylating the hydroxyl group and reducing the azido group of the azidated intermediate to obtain a primary amine intermediate; coupling the primary amine intermediate with benzyl glyoxylate to obtain a backbone intermediate having a protected primary amino group and a benzyloxy carbonyl group; deprotecting the protected primary amino group and the benzyloxy carbonyl group of the backbone intermediate and selectively protecting the backbone intermediate with a protecting group to obtain a protected backbone intermediate; and coupling the protected backbone intermediate with a nucleobase. To prepare left-handed PNA monomer the method comprises: alkylating the hydroxyl group of the dibenzylated intermediate and deprotecting the protected hydroxyl group of the dibenzylated intermediate to obtain an alkoxy intermediate; mesylating and azidatizing the alkoxy intermediate to obtain an azidated intermediate having an azido group; reducing the azido group of the azidated intermediate to obtain a primary amine intermediate; coupling the primary amine intermediate with benzyl glyoxylate to obtain a backbone intermediate having a protected primary amino group and a benzyloxy carbonyl group; deprotecting the protected primary amino group and the benzyloxy carbonyl group of the backbone intermediate and selectively protecting the backbone intermediate with a protecting group to obtain a protected backbone intermediate; and coupling the protected backbone intermediate with a nucleobase. The nucleobase can be any suitable nucleobase, for example and without limitation, nucleobases are independently selected from the group consisting of A, G, C, T, U, unnatural nucleobases or divalent nucleobases. In one aspect, the prepared chiral PNA monomer is optically pure. Any protecting group can substitute for the specifically-listed protecting groups. As indicated above, non-limiting examples of protecting groups include: 9-fluorenylmethyloxy carbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzhydryloxycarbonyl (Bhoc), benzyloxycarbonyl (Cbz), O-nitroveratryloxycarbonyl (Nvoc), benzyl (Bn), allyloxycarbonyl (alloc), trityl (Trt), dimethoxytrityl (DMT), 1-(4,4-dimethyl-2,6-dioxacyclohexylidene)ethyl (Dde), diathiasuccinoyl (Dts), benzothiazole-2-sulfonyl (Bts) and monomethoxytrityl (MMT) groups. In one aspect the protecting group for the amine of the PNA monomer produced by these methods is Fmoc.
(102) PNAs, such as PNA oligomers or polymers, including achiral PNA, RPNA LPNA, and oligomers or polymers combining PNA, RPNA and/or LPNA monomers are prepared from monomers thereof by, e.g., standard peptide synthesis methodologies, for example by solid-phase methods as are broadly-known in the art, involving attaching a peptide to a solid-phase support, followed by repeated cycles of deprotection, washing, coupling and washing for the addition of each specific PNA monomer. Peptide/PNA synthesis methods and services are commercially available, e.g., from Panagene of Daejeon, Korea.
Example 1
Synthesis of LPNA Monomers from D-Amino Acids
(103) LPNA are synthesized from D-amino acids via reductive amination synthesis or a Mitsunobu reaction, according to the reagents and conditions detailed in Scheme 1,
(104) Reagents and conditions: (a) NMM (N-methylmorpholine), IBC (isobutyl chloroformate), DME 4 C., the NaBH.sub.4; (b) Swern oxidation; (c) methyl glycine ester, NMM 4 C., then AcOH (acetic acid), NaBH.sub.3CN; (d) Mitsonobu reaction; (e) CsCO.sub.3, thiophenol, THF (tetrahydrofuran), room temperature; (f) HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), nucleobase, DMF (dimethylformamide), room temperature; (g) 2N NaOH/THF. The nucleobase of step (f) can be any nucleobase, including natural or unnatural nucleobases described herein or elsewhere, for example and without limitation, the divalent nucleobases of WO 2014/169206.
Example 2
Synthesis of (A) RPNA and (B) LPNA Monomers from L-Serine
(105) Both LPNA and RPNA monomers are synthesized from L-serine. The reagents and conditions for this synthesis of RPNA and LPNA are detailed below in Scheme 2 (
Example 3
(106) Herein we report the development of a tight-binding, orthogonal, synthetically versatile and information-interfaced nucleic acid platform, called gamma-peptide nucleic acid (PNA), for programming molecular interactions. The system comprises three molecular entities: the right-handed (RH) and left-handed (LH) helical conformers and a non-helical (NH) domain, with the first two incapable of recognizing each other, and the third capable of recognizing the RH and LH conformers, as well as the natural nucleic acid biopolymers (i.e. DNA and RNA)enabling the storage and translation of genetic information from one system to the next. All three domains are prepared from the same chemical building blocks, with the exception of the stereochemistry or lack thereof at the -backbone that determines if the corresponding oligo adopts an RH, LH or NH motif (
(107) Experimental Procedures
(108) Monomer synthesis.
(109) PNA monomers (1a-d,
(110) Solid-Phase Oligomer Synthesis.
(111) PNA and PNA oligos (Table 3) were synthesized manually on MBHA resin using the standard Boc-solid-phase peptide synthesis procedure. (Dragulescu-Andrasi, et al., A simple -backbone modification preorganizes peptide nucleic acid into a helical structure. J. Am. Chem. Soc. 2006, 128(31):10258-10267). The resulting products were cleaved from the resin and purified by reverse-phase HPLC and characterized by MALDI-TOF. Below are the expected masses of the oligos and those that were found (Table 4).
(112) TABLE-US-00003 TABLE3 SequenceofPNAandPNAoligos - Hel. PNA Sequence ConFigure sense 1 H-.sup.LLys-CCAAC-.sup.LLys-NH.sub.2 Achiral NH 2 H-.sup.LLys-GTTGG-.sup.LLys-NH.sub.2 Achiral NH 3 H-.sup.LLys-CCAAC-.sup.LLys-NH.sub.2 S(L) RH 4 H-.sup.LLys-GTTGG-.sup.LLys-NH.sub.2 S(L) RH 5 H-.sup.LLys-CCAAC-.sup.LLys-NH.sub.2 R(D) LH 6 H-.sup.LLys-GTTGG-.sup.LLys-NH.sub.2 R(D) LH 7 H-.sup.LLys-CCGAC-.sup.LLys-NH.sub.2 R(D) LH 8 H-.sup.LLys-CCCAC-.sup.LLys-NH.sub.2 R(D) LH 9 H-.sup.LLys-CCTAC-.sup.LLys-NH.sub.2 R(D) LH
Bold indicates chiral -Me PNA unit. L and D indicate the stereochemical configurations of the amino acid (alanine) from which these chemical building blocks were prepared. NH: non-helical, RH: right-hand, LH: left-hand.
(113) TABLE-US-00004 TABLE 4 Mass Mass Oligomer calculated found PNA1 1578.64 1575.47 PNA2 1680.69 1677.61 PNA3 1649.78 1647.47 PNA4 1750.82 1749.56 PNA5 1649.78 1646.06 PNA6 1750.82 1747.64 PNA7 1664.77 1660.71 PNA8 1624.75 1621.26 PNA9 1639.76 1635.99 PNA3P 2006.20 2004.48 PNA4P 2108.24 2106.52 PNA5P 2006.20 2001.26 PNA6P 2108.24 2103.60 PNA3C 3636.91 3639.78 PNA4T 3885.12 3887.67 PNA5T 3783.07 3788.84 PNA6C 3738.95 3742.64
(114) CD Measurements.
(115) The samples were prepared in phosphate buffer (10 mM sodium phosphate, 0.1 mM EDTA, 100 mM NaCl, pH=7.2), heated to 90 C. for 5 min and allowed to cool to room temperature overnight. CD experiments were performed on a JASCO J-715 spectropolarimeter using a quartz cuvette with a 1-cm path length. The samples were scanned from 320 to 200 nm at the rate of 100 nm/min and for a total of 15 scans for each run. All spectra were smoothed using five-point adjacent averaging and the baseline (buffer) spectrum was subtracted using Origin software.
(116) UV-Melting Experiments.
(117) The samples were prepared in phosphate buffer (10 mM sodium phosphate, 0.1 mM EDTA, 100 mM NaCl, pH=7.2). Both the heating and cooling runs were performed. UV-melting curves were collected on a Varian Cary 300 Bio UV-Vis spectrometer equipped with a thermoelectrically controlled multi-cell holder. UV-absorbance was collected at 260 nm as a function of temperature from 5 to 90 C. and then from 90 to 5 C. at the rate of 1 C. per min. The T.sub.ms were determined by taking first derivative of the melting curves.
(118) Thermodynamic Analysis.
(119) The duplexes were prepared at 2.5, 5, 10, 15, 20, and 25 M strand concentrations each in phosphate buffer. UV-melting curves were recorded as described above, and the T.sub.ms were determined by taking the first derivatives of the melting curves. A plot ln(C.sub.t) vs. 1/T.sub.m was made, where C.sub.t is the total strand concentration and T.sub.m is the melting transition in Kelvin. From the plot a line fit was made, where the slope equals ((n1)R)/H (n=2 and R=8.314 J/mol K) and the y-intercept equals (S((n1)*R*ln 2n))/H (n=2 and R=8.314 J/mol K). AG was determined using the equation G=HTS, where T=298 K (Tables 5 and 6).
(120) TABLE-US-00005 TABLE 5 Total Temp. Duplex Conc. (M) Conc. (M) ( C.) ln Conc. 1/T (K1) PNA3-PNA4 2.50E06 5.00E06 50 12.21 3.10E03 PNA3-PNA4 5.00E06 1.00E05 53 11.51 3.07E03 PNA3-PNA4 1.00E05 2.00E05 57 10.82 3.03E03 PNA3-PNA4 1.50E05 3.00E05 58 10.41 3.02E03 PNA3-PNA4 2.00E05 4.00E05 59 10.13 3.01E03 PNA3-PNA4 2.50E05 5.00E05 60 9.90 3.00E03 PNA5-PNA6 2.50E06 5.00E06 50 12.21 3.10E03 PNA5-PNA6 5.00E06 1.00E05 54 11.51 3.06E03 PNA5-PNA6 1.00E05 2.00E05 56 10.82 3.04E03 PNA5-PNA6 1.50E05 3.00E05 57 10.41 3.03E03 PNA5-PNA6 2.00E05 4.00E05 58 10.13 3.02E03 PNA5-PNA6 2.50E05 5.00E05 59 9.90 3.01E03
(121) TABLE-US-00006 TABLE 6 Duplex y-Intercept Slope PNA3-PNA4 2.60E03 4.05E05 PNA5-PNA6 2.67E03 3.41E05
(122) Fluorescence Measurements.
(123) The samples were prepared in phosphate buffer, heated to 90 C. for 5 min and allowed to cool to room temperature overnight. Fluorescent profiles were recorded on a Varian Cary Eclipse fluorescence spectrometer using a quartz cuvette with 1-cm path length. The samples were excited at 344 nm, and the fluorescent emissions were scanned from 360 to 600 nm. All spectra were smoothed using five-point adjacent averaging and the baseline (buffer) spectrum was subtracted using Origin software.
(124) Polystyrene Bead Assembly.
(125) Streptavidin-coated polystyrene microspheres were purchased from Spherotech. Since the microspheres were large, they were transferred using micropipettes whose plastic tips were cut to create a larger opening. Before the microspheres were used, they were washed. This was done by transferring 100 L of the slurry solution to an Eppendorf tube, centrifuging the mixture and removing the top buffer layer. The microspheres were then resuspended in 300 L of sodium phosphate buffer and vortexed. The sample was centrifuged and the top buffer layer was removed. The re-suspension, centrifuging and buffer removal (wash cycle) was repeated three times. Then 50 L of PNA (20 M solution) was added to the microsphere mixture and gently place on a shaker for 1.5 hrs to ensure that the biotin-containing PNA oligos were bound to the streptavidin on the microspheres' surface. The microspheres were washed five times to ensure that all unbound biotin-PNA was removed. At this point they should be pink or yellow, depending on the dye used. The microspheres were then resuspended in 400 L of sodium phosphate buffer. This process was done to make 10-PNA4T (RH), 10-PNA6C (LH), 2-PNA3C (RH), and 2-PNA5T (LH). PNA without biotin (but with dye) was also mixed with the microspheres and then washed to determine if PNA itself was interacting with the streptavidin. In this case we did not see any color change in the microspheres, indicating that PNA bound to the microspheres occurred through biotin-streptavidin interaction.
(126) Self-Assembly of Polystyrene Microspheres.
(127) Various combinations of microsphere solutions were prepared and imaged in DIC and fluorescent modes using Olympus IX81 microscope at 40 magnification. Below is a representative of sample preparation. 20 L of 10-PNA4T (RH) and 500 L of the sodium phosphate buffer were placed in a well of the cell culture plate and allowed to settle for 30 min at room temperature, after which point 20 L of 2-PNA3C (RH) was gently added and the mixture was placed on the countertop for 1 hr. At this point, most of the beads settled at the bottom of the well. The plate was transferred to the microscope, and DIC and fluorescent images were taken.
(128) Results and Discussion
(129) Rationale.
(130) Peptide nucleic acid (PNA) has been around for more than two decades, developed by Nielsen and coworkers (Nielsen, P. E., et al., Science 1991, 254, 1497) in 1991 in which the natural sugar phosphodiester backbone was replaced by achiral N-(2-aminoethyl)glycine units (see,
(131) Conformational Analysis.
(132) To determine the effect of -backbone chirality on the conformation of PNA, we measured the CD spectra of PNA1 through 6, individually and after hybridization to their partner strands containing matching sequence and helical sense. Note that PNA3 and 4 contained chemical building blocks with (S)-Me and PNA5 through 9 with (R)-Me at the -backbone. As such we expected the first set to adopt an RH helical motif, as demonstrated in our earlier study (Dragulescu-Andrasi, A., et al., J. Am. Chem. Soc. 2006, 128, 10258; Rapireddy, S., et al., J. Am. Chem. Soc. 2007, 129, 15596), while the helical sense of the latter set was unknown and needed to be determined. The stereochemistry of the terminal lysine residue has no bearing on the conformation or helical sense of PNA as an individual strand (
(133) As expected, no CD signals were observed for PNA1 or 2 in the nucleobase absorption regions (200-320 nm), indicating that in the unhybridized (single-stranded) state PNA does not have a defined helical conformation (
(134) An interesting finding is in comparison of the CD spectra of the individual strands (
(135) Thermal and Thermodynamic Stabilities of the Conformationally-Matched Homoduplexes.
(136) UV-melting experiments were conducted to determine the effect of -backbone modifications on the thermal stability of PNA-PNA duplexes. Our results showed that the melting profiles of the conformationally-matched PNA-PNA duplexes (PN3-PNA4 and PNA5-PNA6) are nearly identical to each other (
(137) Sequence Specificity.
(138) Next, we assessed the ability of PNA to discriminate between closely related sequences. Comparison of the melting profiles of the single-base mismatched duplexes to that of the perfect-match revealed that the destabilization (T.sub.m) is at least 20 C. (Table 7 and
(139) TABLE-US-00007 TABLE 7 Tms of matched and single-base mismatched duplexes Duplex T.sub.m ( C.) T.sub.m ( C.) PNA5-PNA6 54 PNA7-PNA6 (G<>T) 34 20 PNA8-PNA6 (C<>T) ND PNA9-PNA6 (T<>T) 27 27
(140) Recognition Orthogonality.
(141) The opposing helical preference of PNA oligos, RH for PNA3 and 4 and LH for PNA5 and 6, suggested that they might not be able to hybridize to one another despite the sequence complementarity. Such an inherent property would be valuable, if it could be demonstrated, for programming molecular assembly because of the added dimension in recognition. To determine if recognition orthogonality was in play between RH and LH, we measured the thermal stabilities of the conformationally mismatched (PNA3-PNA6 and PNA4-PNA5) and compared them to that of the matched PNA-PNA homoduplexes (PNA3-PNA4 and PNA5-PNA6). Since the melting profiles of the homoduplexes were shown to be nearly identical to each other (
(142) To independently verify the recognition orthogonality of the conformationally-mismatched PNA oligos, we performed fluorescent experiments using pyrene as a reporter probe. Pyrene has been used as a fluorescent marker to investigate the conformational arrangements of proteins and nucleic acids, where formation or disruption of the pyrene excimer is indicative of folding or denaturation (Sahoo, D., et al., Biochemistry 2000, 39, 6594; Yang, C. J., et al., Proc. Nat. Acad. Sci. USA 2005, 102, 17278; Oh, K. J., et al., Nuc. Acid Res. 2006, 34, 152). When excited at 344 nm, the pyrene monomers emit fluorescent signals at 380 and 400 nm, but when they are stacked with each other to form dimers, their excitation results in the formation of excimers which emit at 485 nm. This 100 nm red-shift in the fluorescent emission provides a convenient means for monitoring the on- and off-state of molecular interactions. By attaching pyrene to the terminal regions of the complementary PNA pairs, one at the C and the other at the N-terminus (
(143) The samples were prepared by mixing equimolar concentrations of the various PNA pairs, conformationally-matched as well as mismatched, and annealed at 95 C. for 5 minutes. The mixtures were excited at 344 nm and the fluorescent emissions were recorded from 340 to 600 nm at room temperature. As expected, the samples with complementary sequence and conformationally matched pairs (PNA3P-PNA4P and PNA5P-PNA6P) showed distinct pyrene excimer emissions at 485 nm, with residual monomer emissions at 480 and 400 nm (
(144) Hybridization of RH- and LH-PNA to RNA and DNA.
(145) Next, we assessed the ability of RH- and LH-PNA to hybridize to complementary DNA or RNA strand. UV-melting data revealed that, similar to the observations made with the PNA-PNA homoduplexes, RH-PNA oligos (PNA3 and PNA4) were able to hybridize to complementary DNA (
(146) Dual Recognition of PNA with RH- and LH-PNA.
(147) We have demonstrated that RH- and LH-PNA oligos were unable to hybridize to each other and neither were LH-PNAs to complementary RNA or DNA strands, due to conformational mismatch. Since PNA is achiral and does not have a well-defined conformation, we suspected that it might be able to hybridize to LH as well as RH-PNA. Such a capability is valuable for translating the genetic information encoded in one conformer to another.
(148) Molecular Self-Assembly.
(149) To demonstrate the feasibility and recognition orthogonality of the pentameric PNA system in organizing molecular self-assembly, we employed two sets of polystyrene beads, 2 and 10 m in size, with the surface coated with PNA via biotin-streptavidin binding (
CONCLUSIONS
(150) In summary we have demonstrated that PNA, which, as an individual strand, does not have a well-defined conformation in solution, can be preorganized into a RH or LH helical motif simply by installing an appropriate stereogenic center at the -backbone. LH- and RH-PNA oligos hybridize to their partner strands containing complementary sequence and matching helical sense; however, they do not cross-hybridize with one another. Binding occurs with unusually high affinity and sequence selectivity, presumably through the key and lock motif with minimal conformational rearrangement as the result of backbone preorganization. Recognition modules, as short as 5 nts in length, can be used to organize and assemble micro-particles with high level of recognition orthogonality. Likewise, due to conformational mismatch, LH-PNA oligos are unable to hybridize to complementary DNA or RNA strands. The recognition orthogonality of LH- and RH-PNA can be interfaced with achiral PNA as well as with DNA and RNA, providing an all-in-one nucleic acid platform for programming molecular interactions and assembly.
(151) The utility of PNA in molecular self-assembly and programming chemical reactivity has been demonstrated by Liu et al., Seitz et al. and Winssinger (Kleiner, R. E.; et al., J. Am. Chem. Soc. 2008, 130, 4646; Michaelis, J., et al., Org. Biomol. Chem. 2014, 12, 2812; and Winssinger, N., Chimia 2013, 67, 340). The added dimension in recognition orthogonality, combined with the superior binding affinity and sequence selectivity along with the ease of chemical synthesis and functional group diversification will certainly expand the utility of nucleic acid-based molecular engineering and computing, both in vitro and in vivo. The relatively small size of the recognition module (5 to 8 nts in length) should be relatively easy and cost-effective to synthesize in high-throughput and to scale-up. Moreover, the reduction in size should make cell delivery more manageable and efficient, in comparison to the longer traditional antisense reagents, when functionalized with the appropriate chemical groups or employing an appropriate delivery vehicle. In addition to the LH and RH conformational orthogonality, another dimension of recognition orthogonality that is implemented is base-pairing. Inclusion of unnatural base-pairs, such as isoC and isoG and among others (Piccirilli, J. A., et al. Nature 2000, 343, 33 and Hirao, I., et al., Acc. Chem. Res. 2012, 45, 2055), that do not recognize the natural nucleobases expands the recognition repertoire of such a system further, providing greater flexibility and level of orthogonality in programming molecular interactionfor organization and assembly of materials as well as for molecular computing.
Example 4
(152) This Example provides a rapid and convenient means for diagnosing genetic and infectious diseases, in a laboratory setting as well as in the field. The present detection method has four components: detection of the nucleic acid target; maintenance and restoration of the signal strength; conversion of the detection signal from a right-handed to left-handed helical sense to minimize cross-interference with endogenous genetic materials; and amplification and detection of the signal.
(153) The methods exemplified by this example provide rapid, on-site nucleic acid diagnostics. This is accomplished by employing a series of molecular logic gates to detect the analyte, transmit and amplify the signal in-situ, in the presence of endogenous genetic materials without the use of enzyme or need for background noise washing prior to detection. The new methodology takes advantage of the conformational organization of -peptide nucleic acid (PNA), a nucleic acid mimic that can be directed to fold into either a right-handed or left-handed helix depending on the stereochemistry at the -backbone, and unnatural nucleobases as a means for target detection, signal processing, and amplification.
(154) Like polymerase chain reaction (PCR), reverse-transcriptase/PCR (RT-PCR), and isothermal amplification, detection of nucleic acids by the present invention, I-NADSA, is highly sensitive. However, unlike the established methods, which are labor intensive and time-consuming and prone to cross-contamination and generation of false positives, the present invention is more robust and performed in-situ in the primary specimen without the use of enzyme or concern for background noise. Since the amplification step occurs through a cascade hybridization chain reaction (HCR) free of enzyme, detections of DNA and RNA are carried out the same way, without the need for an additional reverse transcription step for the latter case.
(155) The method exemplified by this example provides several distinctive advantages over existing nucleic acid-based detection methods in terms of speed, sensitivity, specificity, cost, versatility, and portability.
(156) Generally, provided herein is a series of molecular logic gates.
(157) The present invention uses the conformational organization of PNA, a nucleic acid mimic which can be directed to fold into either a right-handed or left-handed helix; and unnatural nucleobases.
(158) The nucleic-acid detection starts with the DNA or RNA target (T1) binding to the toe-hold region of the sensor (1). Branch migration (2) and chain displacement (3) result in the separation of P (same as T2) from S. Due to an increase in backbone flexibility, T2 undergoes further self-splicing (4), resulting in the cleavage of the N-terminal domain (n). Hybridization of T3 (RH) to the toe-hold region of the converter through orthogonal (m-m) nucleobase interactions (5), followed by another round of branch migration (6) and strand displacement (7), results in the release of T4 (LH). Binding of T4 to the first amplifier (8), and then to the second (9), triggers a cascade reaction and the assembly of an extended duplex structure (10). As an example, a FRET-based detection system is shown.
(159) The first component of the present invention, the sensor, includes of a protecting (P) strand and sensing (S) strand hybridized to one another to form a frustrated hairpin structure m with unnatural nucleobases as recognition elements. This is described in
(160) The second component is the restorator, this is detailed in
(161) The next component is the converter, as detailed in
(162)
(163) The present invention has been described with reference to certain exemplary embodiments, dispersible compositions and uses thereof. However, it will be recognized by those of ordinary skill in the art that various substitutions, modifications or combinations of any of the exemplary embodiments may be made without departing from the spirit and scope of the invention. Thus, the invention is not limited by the description of the exemplary embodiments, but rather by the appended claims as originally filed.