The present invention relates to deoxyribozymes and their uses.
Once thought to function primarily as a passive carrier of genetic information, RNA and DNA molecules are now known to be capable of a wide range of functions. For example, nucleic acid molecules called ribozymes (RNA) or deoxyribozymes (DNA) can act as catalysts which accelerate reaction rates millions of times relative to the corresponding nonenzymatic reactions. the structures and functions of catalytic nucleic acids can be allosterically modulated by ligands, and this has been utilized to construct sensors made of RNA and DNA (Tang.J. and Breaker,R.R. (1997) Chem Biol, 4, 453-459; Koizumi,M., Soukup,G.A., Kerr,J.N. and Breaker,R.R. (1999) Nat Struct Biol, 6, 1062-1071). Such sensors consist of a binding domain, which binds the ligand, and a signaling domain, which produces a detectable signal only in the presence of the ligand. Early examples used a self-cleaving ribozyme, and reaction products were detected by PAGE. Later experiments used chemically modified RNA substrates containing a fluorophore at one end and a quencher at the other. Ribozyme-catalyzed cleavage separated the fluorophore from the quencher, and generated a fluorescent signal that could be detected in solution and monitored in real time. Several additional signaling domains made of DNA or RNA have recently been described. These include G-quadruplexes with peroxidase activity which can generate colorimetric or chemiluminescent readouts (Kosman,J. and Juskowiak,B. (2011) Anal Chim Acta, 707, 7-17) and RNA aptamers which enhance the fluorescence of ligands to which they bind (Paige,J.S, Wu,K.Y. and Jaffrey,S.R. (2011) Science, 333, 642-646). These domains typically generate signals 10 to 2000-fold higher than background levels. Limitations include the requirement for expensive chemically modified oligonucleotides or fluorophores that are not commercially available.
The invention relates to a deoxyribozyme which catalyzes a self-phosphorylation reaction in which a phosphate group is transferred from a substrate to the deoxyribozyme. The phosphorylation reaction thus involves cleavage of the phosphate group from the substrate, and its transfer to the deoxyribozyme. The invention further relates to methods of detection of the substrate, or of the deoxyribozyme, or of a ligand such as an oligonucleotide.
The substrate is a compound containing a phosphate functional group and shall be configured so that the cleavage of the phosphate functional group causes a measurable optical signal to be generated. The optical signal may be within the visible light wavelength range, or outside the visible light wavelength range, e.g., in UV or IR wavelengths. Such substrates may be described as chemiluminescent.
The chemiluminescent substrate is preferably a compound of the group of phosphate-stabilized 1,2-dioxetanes. Such substrates are described, e.g., in Bronstein,I., Edwards,B. and Voyta,J.C. (1989) J Biolumin Chemilumin, 4, 99-111; Trayhurn,P., Thomas,M.E., Duncan,J.S., Black,D., Beattie,J.H. and Rayner,D.V. (1995) Biochem Soc Trans, 23, 494S. The compound disclosed in the cited prior art as phosphate-stabilized 1,2-dioxetanes or as chemiluminescent substrates are incorporated herein as examples of chemiluminescent substrates.
1,2-dioxetanes are 4-member ring peroxides which decompose (in the case of our substrates, after cleavage of the stabilizing phosphate group) to form products which are in an excited electronic state. These products may decompose further and emit light.
An example of a chemiluminescent substrate is disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (commercially available under the trade name CDP-Star™). The CDP-Star substrate is known as a substrate of alkaline phosphatase for use in detection of alkaline phosphatase or alkaline phosphatase conjugates, and for detection of non-radioactively labeled nucleic acids in Southern blots, Northern blots, dot blots, colony or plaque hybridizations, gel shift assays. Upon dephosphorylation, the CDP-Star substrate forms meta-stable dioetane phenolate anion which decomposes and emits light at 466 nm.
Another example of a chemiluminescent substrate is disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13.71]decan}-4-yl) phenyl phosphate (commercially available, under the trade name CSPD™). The chemical structure is similar to CDP-Star, and it is used for similar applications.
The deoxyribozyme of the present invention contains or consists of the following nucleotide sequence:
In some embodiments, the deoxyribozyme of the present invention contains or consists of the following nucleotide sequence:
In some embodiments, the deoxyribozyme of the present invention contains or consists of the following nucleotide sequence:
In some embodiments, the deoxyribozyme contains or consists of the following nucleotide sequence:
Generally, nucleotides are selected from adenine nucleotide (A), cytosine nucleotide (C), guanine nucleotide (G), and thymine nucleotide (T). A nucleotide generally contains a base (adenine or cytosine or guanine or thymine) covalently linked to a deoxyribose which is in turn covalently linked to a phosphate.
Preferably, R1 contains 3 to 30 nucleotides, more preferably 3 to 4 nucleotides.
In some embodiments, R1 is a poly-A sequence.
Preferably, R2 contains 3 to 30, more preferably 3 to 10 nucleotides.
In some embodiments, R2 is a poly-A sequence. In some embodiments, R2 is or contains an aptamer or a sequence complementary to an aptamer.
Preferably, R3 contains 3 to 30, more preferably 3 to 10 nucleotides.
In some embodiments, R3 is a poly-A sequence. In some embodiments, R3 is or contains an aptamer or a sequence complementary to an aptamer.
In some embodiments, when R2 is or contains an aptamer, then R3 is or contains a sequence complementary to the aptamer, and when R3 is or contains an aptamer, then R2 is or contains a sequence complementary to the aptamer.
An aptamer is an oligonucleotide sequence that binds to a specific target molecule or target sequence (target molecules and target sequences fall into the group called herein more generally “ligands”). Many aptamer sequences for specific target molecules are known in the art (for example in: Gold,L., Polisky,B., Uhlenbeck,O. and Yarus,M. (1995) Annu Rev Biochem, 64, 763-797; Cho,E.J., Lee,J.W. and Ellington,A.D. (2009) Annu Rev Anal Chem, 2, 241-264). An aptamer for a target sequence is a complementary sequence.
The base pair bp1 and bp2 is preferably formed by canonical T-A, A-T, C-G, and G-C base pairs, more preferably by T-A and A-T base pairs.
The triple helical structure is preferably formed by combinations of canonical C-G:G, T-A:A, and T-A:T base triples, more preferably by C-G:G base triples.
Preferably, each of the helixes 1-3 has the length of 4-10 nucleotides, more preferably 4-7 nucleotides.
In some embodiments, helix 1, helix 2, helix 3 is CCCC, GGGG, GGGG or CCCT, AGGG, GGGT or CTCC, GGAG, GAGG or CCCT, AGGG, GGGA.
In one preferred embodiment, helix1 has the sequence of CCCC, helix 2 has the sequence GGGG, and helix 3 has the sequence GGGG.
Partially disrupted triples can also occur at some positions. These have the ability to form either the Watson-Crick part of the base triple (represented by “-”) or the Hoogsteen part of the base triple (represented by “:”).
When each of the helices 1-3 has the length of 4 nucleotides, they form four triples.
The first triple (positions 13-20-34 in the minimized catalytic core; see
The second triple (positions 14-19-35 in the minimized catalytic core; see
The third triple (positions 15-18-36 in the minimized catalytic core; see
The fourth triple (positions 16-17-37 in the minimized catalytic core; see
Preferably, the deoxyribozyme contains 45 to 160 nucleotides, more preferably 45 to 110 nucleotides in total, even more preferably 80 to 110 nucleotides.
The invention thus provides a deoxyribozyme that phosphorylates itself in the presence of a phosphate group-containing substrate, such as phosphate-stabilized 1,2-dioxetane substrates, to generate an optical signal. By “deoxyribozyme”, it is meant a DNA oligonucleotide which catalyzes a chemical reaction. This meaning includes DNA oligonucleotides with chemical modifications. Deoxyribozymes of the present invention were developed with the aid of a method of artificial evolution called in vitro selection. Catalytic oligonucleotides isolated using in vitro selection typically contain a catalytic core, usually consisting of stems forced by A-T, T-A, G-C, and C-G base pairs, interspersed with sequence elements not required for function.
The catalytic core of the deoxyribozyme described herein has the following features:
This overhang can be extended by the extension R3 with the sequence N0-30 (N=A, C, G, or T).
The deoxyribozyme of the present invention, upon contact with a substrate, can cause the generation of an optical signal. The optical signal is generated by dephosphorylation of the substrate by the deoxyribozyme. This triggers decomposition of the substrate and, in case of the CDP-Star™ and CSPD™ substrates, production of blue light with a wavelength of ˜466 nm by a CIEEL mechanism. The deoxyribozyme can be configured so that it is inactive (inactive state) in the absence of a ligand, but undergoes an allosteric change to the catalytically active structure (active state) in the presence of the ligand. In this way the deoxyribozyme can be used to detect specific ligands. The substrate is as defined herein above.
The present invention further relates to a method of in vitro detection of a chemiluminescent substrate, containing a phosphate functional group, in a sample, containing the steps of:
In this method, it is advantageous when R1 and R2 are not mutually complementary.
In this method, it is advantageous when R1 and R3 are not mutually complementary.
In this method, it is advantageous when R2 and R3 are not mutually complementary.
The present invention also relates to a method of in vitro detection of the deoxyribozyme in a sample, containing the steps of:
The step of causing the generation of the optical signal typically includes providing suitable reaction conditions for the self-phosphorylation reaction of the deoxyribozyme. Such reaction conditions may include, for example, metal ions in the buffer, including potassium, zinc, cerium, lead (e.g., up to 200 mM KCl, 1 mM ZnCl2, 1 μM Ce(SO4)2, and 0.1 μM PbCl2), pH (e.g. 7-8), buffer (e.g. HEPES), temperature (e.g. 20-30° C.), deoxyribozyme concentration (e.g. 1-50 μM or 0.1-10 μM), substrate concentration (e.g. 0.001-1 mM, preferably 0.01-100 μM) and/or ligand concentration (e.g. 1 to 100 μM).
In some preferred embodiments, the deoxyribozyme is also active in a simplified buffer containing HEPES, zinc, and potassium. In such a buffer, light is most efficiently generated over a narrow range of zinc concentrations (between around 0.3 mM and 1.5 mM) and pH values (between around pH 6.9 and 7.7). Potassium slightly increases light production, but is inhibitory at concentrations above about 200 mM. Light production can also be increased by reducing the substrate concentration (˜62.5 μM is optimal) and increasing the deoxyribozyme concentration (about 30 μM is optimal). Rate enhancements of light production (defined as the amount of light produced in the presence of deoxyribozyme divided by the amount of light produced in the absence of deoxyribozyme) can also be increased by using optimized concentrations of buffer components either individually (e.g. up to 200 mM KCl, 1 mM CDP-Star, 30 μM deoxyribozyme, 650 μM ZnCl2, 15% DMSO, and 50 mM HEPES, pH 7.4, column 4 in
The present invention further relates to a method of in vitro detection of a ligand in a sample, containing the steps of:
The ligand may be, for example, an oligonucleotide, a small molecule (e.g., molecular weight up to 2000 g/mol), a metal ion, a protein, or a lipid. The ligand is in fact an analyte detected in a sample.
The ligand binds to the aptamer, thus changing the conformation of the deoxyribozyme from the inactive state to the active state, and the deoxyribozyme in the active state then self-phosphorylates, thus cleaving the phosphate group from the substrate. This triggers a decomposition reaction and the production of blue light by a CIEEL mechanism.
For example, when the ligand is an oligonucleotide, the R2 in the deoxyribozyme may be or contain the sequence or part of the sequence of the ligand (oligonucleotide), and the R3 in the doxyribozyme may be or contain a sequence complementary to the sequence or to part of the sequence of the ligand. The oligonucleotide binds to R3 by Watson-Crick pairing. At sufficiently high concentrations, this prevents formation of the inhibitory interaction between R2 and R3. When the oligonucleotide is bound to R3, the deoxyribozyme is catalytically active and transfers the phosphate group from the substrate to the deoxyribozyme. This triggers a decomposition reaction and the production of blue light by a CIEEL mechanism.
Also when the ligand is a small molecule or a metal ion, or a protein, or a lipid, binding of the ligand to an aptamer suitable for the ligand causes conformational changes in the deoxyribozyme leading to its self-phosphorylation. For example, a sequence complementary to the aptamer can be inserted into R2 and the aptamer can be inserted into R3. In the absence of the ligand of the aptamer, an inhibitory interaction will form between R2 and R3 by Watson-Crick pairing. In the presence of sufficiently high concentrations of the ligand, however, the aptamer will bind the ligand rather than R2. This will prevent formation of the inhibitory interaction between R2 and R3. When the ligand is bound to the aptamer, the deoxyribozyme is catalytically active and transfers the phosphate group from the substrate to the deoxyribozyme. This triggers a decomposition reaction and the production of blue light by a CIEEL mechanism.
The step of causing the generation of the optical signal typically includes providing suitable reaction conditions for the self-phosphorylation reaction of the deoxyribozyme. Such reaction conditions may include, for example, metal ions in the buffer, including potassium, zinc, cerium, lead (e.g., 200 mM KCl, 1 mM ZnCl2, 1 μM Ce(SO4)2, and 0.1 μM PbCl2), pH (e.g. 7-8), buffer (e.g. HEPES), temperature (e.g. 20-30° C.), deoxyribozyme concentration (e.g. 0.1-10 μM), and/or substrate concentration (e.g. 0.001-1 mM), and ligand concentration (e.g. 1 to 100 μM).
Light generated by the deoxyribozyme can be detected using an instrument such as a plate reader, e.g. Tecan Spark plate reader.
Generally, the detection of the light may be qualitative or quantitative. Quantitative detection contains an additional step of determining the amount of the optical signal generated.
The invention also relates to the use of the deoxyribozyme of the present invention as a signaling component of molecular devices in applications such as biosensing and molecular computing. Molecular devices may include molecular sensors or molecular switches, such as deoxyribozymes that only generate light in the presence of a specific ligand such as ATP. Suitable applications of such molecular devices include diagnostics, high-throughput screens, and molecular computing. The deoxyribozyme of the present invention is used in the molecular device as the signaling component causing the generation of an optical signal which indicates the presence of a ligand (e.g., a metal ion, a small molecule, a protein, an oligonucleotide, a lipid), and optionally the molecular device is configured to perform an action upon generation of the said signal.
A second object of the invention relates to the use of this deoxyribozyme in molecular devices that generate a chemiluminescent signal. An example of such a device is an allosterically regulated sensor in which the deoxyribozyme described here (the signaling domain) is covalently linked to an aptamer (the binding domain) that binds a small molecule, oligonucleotide, or protein with high affinity and specificity. An example of such a sensor is shown in
A single-stranded pool with the sequence GGAAGAGATGGCGACN70AGCTGATCCTGATGG (SEQ ID NO. 4) was ordered from IDT and purified by PAGE. 1016 different sequences from this pool were mixed with the blocking oligonucleotide BO1 (CCATCAGGATCAGCT, SEQ ID NO. 5) in water, heated at 65° C. for 2 minutes and cooled at RT for 5 minutes. They were then incubated for 24 hours (round 1-6) or 10 minutes (round 7-9) with the substrate CDP-Star (Roche) as follows: 1 μM DNA pool, 1.5 μM blocking oligonucleotide BO1, 1× selection buffer (50 mM HEPES pH 7.4, 200 mM KCl, 1 mM ZnCl2, 1 μM Ce(SO4)2, 0.1 μM PbCl2), and 1 mM CDP-Star DNA was then precipitated in ethanol, and ligated to a short oligonucleotide with the sequence. GAATTCTAATACGACTCACTATA (SEQ ID NO. 6) using a splint-ligation method, T4 DNA ligase (Jena Bioscience), and a splint oligonucleotide with the sequence GTCGCCATCTCTTCCTATAGTGAGTCGTATTAG (SEQ ID NO. 7). All oligonucleotides were kept at a concentration of 2.5 μM. Molecules were then separated by gel shift using 6% Urea-PAGE, and molecules of the size 120 nt (corresponding to ligated pool members) were excised from the gel, eluted, and precipitated with ethanol. These molecules were then amplified by PCR using Tag Polymerase (Jena Bioscience) and the FWD1 (GAATTCTAATACGACTCACTATA, SEQ ID NO. 6) and REV1 (CCATCAGGATCAGCT, SEQ ID NO. 5) primers. The FWD1 primer contained a single RNA linkage at its 3′ end so the reaction site of the deoxyribozyme could be regenerated. After another ethanol precipitation, the sense strands of the double-stranded molecules were base hydrolyzed, and the two strands were separated using 6% Urea-PAGE to regenerate the single-stranded DNA pool.
A library was generated by randomly mutagenizing the most active deoxyribozyme isolated in the original selection:
After 6 rounds of the reselection the evolved pool was characterized by high-throughput sequencing. The analysis was performed using the Illumina HiSeq System (2×150 bp, paired-end; NGSelect Amplicon product by Eurofins Genomics). Raw reads were processed with the cutadapt tool to remove adapter and primer sequences, perform quality trimming, and filter low quality reads. Paired-end reads were oriented, merged with the program fastq-join, and aligned using Clustal Omega. Data quality was evaluated using the FastQC tool. The secondary structure of the deoxyribozyme was predicted using comparative sequence analysis, which included frequency calculations of all di- and trinucleotide combinations and mutual information analysis (Gutell,R.R., Power,A., Hertz,G.Z., Putz,E.J. and Stormo,G.D. (1992). Nuc Acids Res, 20, 5785-57951) using in-house scripts. Analysis of conservation led to the identification of the consensus sequence 5′ GGAAGA-(26 variable nucleotides)-GAATATCCCC-(18 variable nucleotides)-GGGGAGTGACTTGGGATGGGGG 3 (SEQ ID NO. 14). Detailed information about all tools used is listed in Table 1.
Oligonucleotides were ordered from Sigma-Aldrich and purified by PAGE or HPLC prior to use. Each deoxyribozyme was mixed with either water alone or water and the appropriate blocking oligonucleotide (indicated in Examples 5 and 6), heated at 65° C. for 2 minutes, and cooled at RT for 10 minutes. After that, 5× Selection Buffer and CDP-Star™ were added. Final concentrations were 1 μM deoxyribozyme, 1.5 μM blocking oligonucleotide (when needed), 1× Selection Buffer (50 mM HEPES pH 7.4, 200 mM KCl, 1 mM ZnCl2, 1 M Ce(SO4)2, 0.1 μM PbCl2), and 1 mM CDP-Star™. Reactions were incubated for various times in the dark at RT and ethanol-precipitated. Deoxyribozymes were then ligated to a short oligonucleotide as described in Example 1. Reacted and unreacted molecules were then separated on 6% PAGE gels, and the amount of ligated product was determined using ImageQuant TL software (GE Healthcare LifeSciences).
Sequences of the isolated deoxyribozymes were ordered from Sigma-Aldrich and purified by PAGE or HPLC prior to use. Deoxyribozymes were mixed with either water or water and the appropriate blocking oligonucleotide, heated at 65° C. for 2 minutes, and cooled at RT for 10 minutes. After that, 5× Selection Buffer was added, and samples were transferred to a white half-area 96 well plate (Corning). CDP-Star™ was added, and chemiluminescence was measured for 1 to 24 hours using a Tecan Spark plate reader (Tecan Group). Final concentrations were 1 μM deoxyribozyme, 1.5 μM blocking oligonucleotide (when necessary), 1× Selection Buffer (50 mM HEPES pH 7.4, 200 mM KCl, 1 mM ZnCl2, 1 μM Ce(SO4)2, 0.1 μM PbCl2), and 1 mM CDP-Star™.
Deoxyribozymes from the initial selection were tested as described in Example 4, except using 0.25 mM rather than 1 mM CDP-Star™ substrate. The most active variant was
The ten most abundant deoxyribozymes in the reselection were tested as described in Example 4, except using 0.25 mM rather than 1 mM CDP-Star™ substrate. The most abundant deoxyribozyme was
The second most abundant deoxyribozyme was
The third most abundant deoxyribozyme was
The fourth abundant deoxyribozyme was
The fifth most abundant deoxyribozyme was
The sixth most abundant deoxyribozyme was
The seventh most abundant deoxyribozyme was
The eighth most abundant deoxyribozyme was
The ninth most abundant deoxyribozyme was
The tenth most abundant deoxyribozyme was
To identify the catalytic core of the deoxyribozyme, variable regions in the sequence alignment of deoxyribozymes from the reselection (Example 1) were either deleted or replaced with AAAA linkers. The minimized deoxyribozyme also contained a T to C mutation at position 15 in helix 1 (numbering corresponds to that used in
The sequence of the minimized catalytic core of the deoxyribozyme is
The amount of product in the ligation assay was 34.6% relative to a control oligonucleotide containing a 5′ phosphate. It also generated 128-fold more light than the background reaction in the absence of deoxyribozyme in the light-production assay. The catalytic activities of the variants described in Examples 8-13 are expressed relative to the activity of this construct.
To confirm the triple helix in the secondary structure model of the deoxyribozyme, a series of mutants were constructed in which a proposed base triple involving positions 15, 18, and 36 (numbering corresponds to that used in
The first mutant in the series
The second mutant in the series
The third mutant in the series
The fourth mutant in the series
The fifth mutant in the series
The sixth mutant in the series
The seventh mutant in the series
The eighth mutant in the series
Minimized catalytic cores containing point mutations in unpaired regions (positions 1-11, 22-33, and 38; numbering as in
3 × 10−1
5 × 10−1
The first construct in this series contains the 1T mutation. It has the sequence
The second construct in this series contains the 2T mutation. It has the sequence
The third construct in this series contains the 3C mutation. It has the sequence
The fourth construct in this series contains the 5T mutation. It has the sequence
The fifth construct in this series contains the 6T mutation. It has the sequence
The sixth construct in this series contains the 7T mutation. It has the sequence
The seventh construct in this series contains the 7A mutation. It has the sequence
The eighth construct in this series contains the 8C mutation. It has the sequence
The ninth construct in this series contains the 8G mutation. It has the sequence
The tenth construct in this series contains the 8T mutation. It has the sequence
The eleventh construct in this series contains the 9T mutation. It has the sequence
The twelfth construct in this series contains the 9G mutation. It has the sequence
The thirteenth construct in this series contains the 10A mutation. It has the sequence
The fourteenth construct in this series contains the 10C mutation. It has the sequence
The fifteenth construct in this series contains the 10G mutation. It has the sequence
The sixteenth construct in this series contains the 11T mutation. It has the sequence
The seventeenth construct in this series contains the 11G mutation. It has the sequence
The eighteenth construct in this series contains the 22A mutation. It has the sequence
The nineteenth construct in this series contains the 27C mutation. It has the sequence
The twentieth construct in this series contains the 27G mutation. It has the sequence
The twenty first construct in this series contains the 33G mutation. It has the sequence
The twenty second construct in this series contains the 33C mutation. It has the sequence
The twenty third construct in this series contains the 38T mutation. It has the sequence
The twenty fourth construct in this series contains the 38A mutation. It has the sequence
The twenty fifth construct in this series contains the 38C mutation. It has the sequence
Minimized catalytic cores containing different combinations of canonical base pairs (at the 12-21 base pair) and base triples (at the 13-20-34, 14-19-35, 15-18-36, and 16-17-37 base triples; numbering as in
The first construct in this series contains T at position 12 and A at position 21. It has the sequence
The second construct in this series contains A at position 12 and T at position 21. It has the sequence
The third construct in this series contains C at position 12 and G at position 21. It has the sequence
The fourth construct in this series contains G at position 12 and C at position 21. It has the sequence
The fifth construct in this series contains C-C:G base triples at positions 13-20-34, 14-19-35, 15-18-36, and 16-17-37. It has the sequence
The sixth construct in this series contains a T-A:A base triple at position 13-20-34 and C-G:G triples at the other positions in the triple helix. It has the sequence
The seventh construct in this series contains a T-A:T base triple at position 13-20-34 and C-G:G triples at the other positions in the triple helix. It has the sequence
The eighth construct in this series contains a T-A:A base triple at position 14-19-35 and C-G:G triples at the other positions in the triple helix. It has the sequence
The ninth construct in this series contains a T-A:T base triple at position 14-19-35 and C-G:G triples at the other positions in the triple helix. It has the sequence
The tenth construct in this series contains a T-A:A base triple at position 15-18-36 and C-G:G triples at the other positions in the triple helix. It has the sequence
The eleventh construct in this series contains a T-A:T base triple at position 15-18-36 and C-G:G triples at the other positions in the triple helix. It has the sequence
The twelfth construct in this series contains a T-A:A base triple at position 16-17-37 and C-G:G triples at the other positions in the triple helix. It has the sequence
The thirteenth construct in this series contains a T-A:T base triple at position 16-17-37 and C-G:G triples at the other positions in the triple helix. It has the sequence
The fourteenth construct in this series contains a C-G:G base triple at position 13-20-34 and T-A:A base triples at position 14-19-35, 15-18-36, and 16-17-37. It has the sequence
The fifteenth construct in this series contains a C-G:G base triple at position 13-20-34 and T-A:T base triples at position 14-19-35, 15-18-36, and 16-17-37. It has the sequence
The sixteenth construct in this series contains T-A:A base triples at all positions in the triple helix. It has the sequence
The seventeenth construct in this series contains T-A:T base triples at all positions in the triple helix. It has the sequence
The eighteenth construct in this series contains a deleted triple at position 16-17-37 and C-G:G triples at the other positions in the triple helix. It has the sequence
The nineteenth construct in this series contains deleted triples at positions 16-17-37 and 15-18-36, and C-G:G triples at the other positions in the triple helix. It has the sequence
The twentieth construct in this series contains deleted triples at positions 16-17-37, 15-18-36, and 14-19-35, and a C:G:G triple at position 13-20-34 in the triple helix. it has the sequence
Minimized catalytic cores containing different partially or completely disrupted triples at the 13-20-34, 14-19-35, 15-18-36, and 16-17-37 base triples (numbering as in
The first construct in the series contains an A G:G triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a G G:G triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a T G:G triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a A A:A triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a C A:A triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a G A:A triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a A A:T triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a C A:T triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a G A:T triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a C-G A triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a C-G T triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a T-A G triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a T G T triple at position 13-20-34 in the triple. helix. It has the sequence
The next construct in the series contains a G G C triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a C C G triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a C A G triple at position 13-20-34 in the triple helix. It has the sequence
The next construct in the series contains a A G:G triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a G G:G triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a T G:G triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a A A:A triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a C A:A triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a G A:A triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a A A:T triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a C A:T triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a G A:T triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a C-G A triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a C-G T triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a T-A G triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a T G T triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a G G C triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a C C G triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a C A G triple at position 14-19-35 in the triple helix. It has the sequence
The next construct in the series contains a A G:G triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a G G:G triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a T G:G triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a A A:A triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a C A:A triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a G A:A triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a A A:T triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a C A:T triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a G A:T triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a C-G A triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a C-G T triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a T-A G triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a T G T triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a G G C triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a C C G triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a C A G triple at position 15-18-36 in the triple helix. It has the sequence
The next construct in the series contains a A G:G triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a G G:G triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a T G:G triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a A A:A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a C A:A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a G A:A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a A A:T triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a C A:T triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a G A:T triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a C-G A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a C-G T triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a T-A G triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a A-T A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a G-C A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a T G T triple at position 16-17-37 in the triple helix. It has
The next construct in the series contains a G T A triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a G G C triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a C C G triple at position 16-17-37 in the triple helix. It has the sequence
The next construct in the series contains a C A G triple at position 16-17-37 in the triple helix. It has the sequence
The 50 most abundant minimized catalytic cores of deoxyribozymes in the reselection (cores 1-50) were tested as described in Example 4, except using 0.25 mM rather than 1 mM CDP-Star™ substrate. These correspond to sequences in which R1 was replaced by AAAA, R2 was replaced by AAAA, and R3 was deleted. Core 1 (SEQ ID NO. 25) is already described in Example 7, Cores 2 (SEQ ID NO:26) and 3 (SEQ ID NO:32) are already described in Example 8, Cores 8 (SEQ ID NO:40), 12 (SEQ ID NO:48), 19 (SEQ ID NO:55), 20 (SEQ ID NO:41), 21 (SEQ ID NO:42), 24 (SEQ ID NO:57), 39 (SEQ ID NO:47), 40 (SEQ ID NO:37), 42 (SEQ ID NO:46), and 48 (SEQ ID NO:56) are already described m Example 9. cores 16 (SEQ ID NO:66), 32 (SEQ ID NO:67), and 46 (SEQ ID NO:63) are already described in Example 10, and Cores 5 (SEQ ID NO:92) 6 (SEQ ID NO:108), 34 (SEQ ID NO:101), 41 (SEQ ID NO:94) are already described in Example 11. The remaining cores are listed here.
The sequence of core 4 is
The sequence of core 7 is
The sequence of core 9 is
The sequence of core 10 is
The sequence of core 11 is
The sequence of core 13 is
The sequence of core 14 is
The sequence of core 15 is
The sequence of core 17 is
The sequence of core 18 is
The sequence of core 22 is
The sequence of core 23 is
The sequence of core 25 is
The sequence of core 26 is
The sequence of core 27 is
The sequence of core 28 is
The sequence of core 29 is
The sequence of core 30 is
The sequence of core 31 is
The sequence of core 33 is
The sequence of core 33 is
The sequence of core 36 is
The sequence of core 37 is
The sequence of core 38 is
The sequence of core 43 is
The sequence of core 44 is
The sequence of core 45 is
The sequence of core 47 is
The sequence of core 49 is
The sequence of core 50 is
Deletion analysis (described in Example 7) showed that R1 (originally 26 nucleotides) could be replaced by AAAA, that R1 (originally 18 nucleotides) could be replaced by AAAA, and R3 (originally 18 nucleotides) could be deleted (see
The first deoxyribozyme in the series
The second deoxyribozyme in the series
The third deoxyribozyme in the series
The fourth deoxyribozyme in the series
The fifth deoxyribozyme in the series
The sixth deoxyribozyme in the series
The seventh deoxyribozyme in the series
The eighth deoxyribozyme in the series
The ninth deoxyribozyme in the series
To further characterize deoxyribozyme insertion sites, five different variants of each of the nine R1-R2-R3 architectures described in Example 13 were generated in which arbitrary sequences were inserted at R1, R2, and/or R3. In addition, five different variants of each of two new R1-R2-R3 architectures were generated. See
The first deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
The next deoxyribozyme in the series
To generate a version of this deoxyribozyme that can sense oligonucleotides with specific sequences, we inserted part of the sequence of the target oligonucleotide into R2, and the reverse complement of the full target oligonucleotide into R3. In the absence of the target, R2 and R3 form a helix by Watson-Clack interactions that inhibits the deoxyribozyme. In the presence of the target, however, the inhibitory helix cannot form because R3 binds to the target rather than R2. This results in catalytic activity and the production of light. Deoxyribozymes were tested as described, in Example 4 (except using 0.25 mM rather than 1 mM CDP-Star™ substrate) in the presence of either 0 μM or 10 μM oligonucleotide ligand (=target).
The first sensor we generated
The second sensor we generated
The third sensor we generated
The fourth sensor we generated
The fifth sensor we generated
A deoxyribozyme with the sequence SEQ ID NO. 25 was tested as described in Example 4 except for the following changes.
In
In
In
In
In
In
All other reactions in
The y axis shows the rate enhancement of light production (the amount of light produced in the presence of deoxyribozyme divided by the amount of light produced in the absence of deoxyribozyme).
For all panels, points show the average of at least three experiments.
Number | Date | Country | Kind |
---|---|---|---|
21156992.6 | Feb 2021 | EP | regional |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/CZ2022/050001 | 1/12/2022 | WO |
Number | Date | Country | |
---|---|---|---|
20240132941 A1 | Apr 2024 | US |