Fine-adjusted super-specific nucleic acid hybridization probe

Abstract

The present disclosure relates to finely modulated ultra-specific nucleic acid hybridization probes. The present invention provides compositions and methods for highly specific nucleic acid probes and primers. The probe system includes a complementary strand and a protective strand to partially form a double-stranded probe. The reaction standard free energy ([delta] G DEG rxn = [delta] G DEG t-TC-[delta] G DEG nh-PC + ([delta] G DEG v-TC-[delta] G DEG h-PC)) of hybridization between the probe and the target nucleic acid determined by formula 1 is about-4 kcal / mol to + 4 kcal / mol. Alternatively, the reaction standard free energy of hybridization between the probe and the target nucleic acid determined by Formula 1 is within 5 kcal / mol of the standard free energy determined by Formula 2 (-R [tau] ln (([P] 0-[C] 0) / [C] 0)), the term [P] 0 of Formula 2 being equal to the concentration of the protective strand, and the term [C] 0 of Formula 2 being equal to the concentration of the complementary strand. In addition, the present invention provides a method for the immediate fine modulation of a reaction using the probe of the present invention.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201480075577.7.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 61 / 916,321, filed December 16, 2013, which is incorporated herein by reference in its entirety. background

[0004] Tiny differences in DNA and RNA sequences can lead to vast differences in health. For example, a single base change in a bacterial genome can lead to antibiotic resistance, and a single base change in the human genome can lead to cancer remission. With the maturation of genomics and the subsequent discovery of numerous nucleic acid biomarker sequences and molecules, there is a strong demand from the biotechnology industry for reliable, robust, inexpensive, and accurate nucleic acid assays that can distinguish single base changes. Enzyme-based methods for differentiating nucleic acid sequences are difficult to integrate with various technologies because enzymes require specific temperature and buffering conditions.

[0005] Enzyme-free hybridization techniques that ensure high specificity of nucleic acids and their complements have traditionally relied on optimizing melting temperatures, which are difficult to predict and control precisely. Recently, toehold hybridization probes have been shown to robustly recognize single-base alterations in nucleic acid sequences over a wide temperature and salinity range. These probes are designed to operate at the standard reaction free energy (ΔG). o rxn They react with their intended targets at near-zero levels, resulting in a hybridization rate of nearly 50%. Even a single nucleotide change in the target can cause it to bind to the probe with a significantly lower hybridization rate (median 2%).

[0006] To achieve ΔG o rxn With a binding energy of ≈0, these probes balance the binding energy between the target-specific "sticky end" region and the target-non-homologous "balance" region. DNA probes have been experimentally proven to robustly distinguish DNA targets, and RNA probes have been experimentally proven to robustly distinguish RNA targets.

[0007] However, these probes are subject to several limitations. For example, when the probe and target are different types—such as when a DNA probe is designed to be specific to an RNA target, a 2'-O-methylRNA probe is designed to bind to an RNA target, and an LNA probe is designed to specifically bind to a DNA target—the thermodynamic differences in hybridization between different types of nucleic acid molecules can lead to poor probe design with low specificity or low sensitivity. Additionally, the relatively high thermodynamic binding strength of individual base pairs / stacks particularly hinders the reaction ΔG. o rxn The fine-tuning of this process limits the adjustability of the trade-off between specificity and sensitivity in probe systems. Furthermore, the publicly known thermodynamic parameters of DNA and RNA hybridization are known to be incomplete and / or erroneous under certain conditions. Computer-designed probe systems can have parameters consistent with the calculated ΔG. o rxn Significantly different real ΔG o rxn Without a method for fine-tuning probe performance, iterative trial and error must be used to achieve the desired ΔG. o rxn The optimal probe design. Invention Overview

[0008] According to certain examples, this invention provides highly specific nucleic acid hybridization probe systems that reliably distinguish single-base alterations in target nucleic acids. Compared to existing technologies, the probe systems described in this invention offer the advantages of (1) reliably detecting DNA, RNA, and modified nucleic acid targets using DNA, RNA, and other nucleic acid probes; and (2) enabling fine-tuning of the trade-off between sensitivity and specificity. The compositions and methods of this invention can be used as tools for molecular cancer diagnosis, infectious disease diagnosis, food safety diagnosis, and research discovery based on DNA and RNA detection and quantification.

[0009] In one example, a composition is provided for selectively interacting with a target nucleic acid molecule. The composition comprises a first nucleic acid strand containing a first region, a second region, and a third region at a first concentration, and a second nucleic acid strand containing a fourth region and a fifth region at a second concentration. The target nucleic acid includes nucleotide sequences of a sixth and a seventh region, which are at least partially complementary to the nucleotide sequences of the first and second regions, even if incompletely. The first and second concentrations result in an interaction between the target nucleic acid and the composition having a standard free energy within 5 kcal / mol of Equation 1[ΔG], determined by Equation 2(-Rτln(([P]0-[C]0) / [C]0)]). o rxn =ΔG o t-TC -ΔG onh-PC +(ΔG o v-TC -ΔG o h-PC The standard free energy (ΔG) determined by )] o rxn In Equation 2, [P]0 equals the second concentration, [C]0 equals the first concentration, R equals the universal gas constant 8.314 J / mol·K, and τ equals the Kelvin temperature. In this example, ΔG in Equation 1... o t-TC The standard free energy representing the hybridization between region 6 and region 1; ΔG in Equation 1 o nh-PC This represents the free energy of hybridization between the fifth and third regions; ΔG in Equation 1 o v-TC The standard free energy representing the hybridization between the seventh and second regions; ΔG in Equation 1 o h-PC This represents the standard free energy of the hybridization between the fourth and second regions. Calculate ΔG. o The method for determining the value will be detailed later in the specification. In some cases, the concentration of the target nucleic acid is less than the first concentration. In other cases, the concentration of the target nucleic acid is greater than or equal to the first concentration.

[0010] In another example, the sequences of the first, second, third, fourth, fifth, sixth, and seventh regions enable the interaction between the target nucleic acid and the composition to have Equation 1 [ΔG]. o rxn =ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC The standard free energy (ΔG) determined by the range of approximately -4 kcal / mol to +4 kcal / mol is... o rxn ), where [ΔG o t-TC -ΔG o nh-PC It is not between -1 kcal / mol and +1 kcal / mol. In other examples, ΔG o t-TC and ΔG o nh-PC The values ​​are all within 10% of each other.

[0011] In another example, the target nucleic acid also includes an eighth region adjacent to the seventh region, the nucleotide sequence of the eighth region is not complementary to the nucleotide sequence of the third region, and the number of aligned nucleotide pairs between the eighth and third regions is less than 50% under equilibrium.

[0012] In another example, a method for generating a nucleic acid probe is provided. The method includes the steps of: selecting a target nucleotide sequence in a nucleic acid molecule, the target nucleotide sequence comprising a sixth nucleotide subsequence and a seventh nucleotide subsequence; selecting a first nucleotide sequence comprising a first nucleotide subsequence, a second nucleotide subsequence, and a third nucleotide subsequence; and selecting a second nucleotide sequence comprising a fourth nucleotide subsequence and a fifth nucleotide subsequence. In this example, the selection of the first, second, and target nucleotide sequences is based on having Equation 1 [ΔG] o rxn =ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC The interaction between approximately -4 kcal / mol and approximately +4 kcal / mol, determined by the standard free energy of Equation 1, is ΔG. o t-TC The standard free energy of hybridization between region 6 and region 1 is represented by ΔG in Equation 1. o nh-PC The free energy of hybridization between the fifth and third regions is represented by ΔG in Equation 1. o v-TC The standard free energy represents the hybridization energy between the seventh and second regions, and ΔG in Equation 1. o h-PC The standard free energy represents the hybridization free energy between the fourth region and the second region. The method also includes the steps of synthesizing a first nucleotide chain containing a first nucleotide sequence and a second nucleotide chain containing a second nucleotide sequence.

[0013] In addition to selecting the relevant nucleotide sequence based on Equation 1, the method may alternatively or further include selecting first and second concentrations such that the standard free energy determined by Equation 2 (-Rτln(([P]0-[C]0) / [C]0)) is equal to the standard free energy determined by Equation 1 (ΔG). o rxnThe standard free energy determined by Equation 1 is within 5 kcal / mol, where [C]0 and [P]0 in Equation 2 represent the predetermined concentrations of the first and second nucleotide chains, respectively, R equals the universal gas constant 8.314 J / mol·K, and τ equals the Kelvin temperature. In one example, if the standard free energy determined by Equation 1 is not within 5 kcal / mol of the standard free energy determined by Equation 2, the predetermined concentration of at least one of the first or second nucleic acid chains can be improved until it meets the condition. Alternatively, optimization can be performed by repeating the steps of this method and selecting a modified nucleotide sequence to meet the desired free energy condition.

[0014] A method is provided for identifying the presence or amount of a nucleic acid molecule having a target nucleotide sequence in a sample. The method includes applying a probe to a sample that may contain the target nucleic acid molecule and performing a hybridization reaction at a temperature of about 4°C to about 75°C, about 25°C to about 70°C, or about 37°C to about 65°C, or any temperature between these temperatures, to allow hybridization of the probe with the target nucleic acid molecule in the presence of the target nucleic acid molecule in the sample. In this example, the probe includes a first nucleic acid strand and a second nucleic acid strand. The first nucleic acid strand contains a first region, a second region, and a third region, wherein the first region has a nucleotide sequence complementary to a sixth region of the target nucleic acid molecule, and the second region has a nucleotide sequence complementary to a seventh region of the target nucleic acid molecule. The second nucleic acid strand contains a fourth region and a fifth region, wherein the fourth region has a nucleotide sequence complementary to the second region of the target nucleic acid molecule, and the fifth region has a nucleotide sequence complementary to the third region of the target nucleic acid molecule. In one example, the target nucleic acid molecule is RNA.

[0015] A method for selectively amplifying a target nucleic acid sequence from a sample, the method comprising applying a probe as an enzyme primer to a mixture containing the sample, a DNA or RNA polymerase, and a mixture of nucleotide triphosphates. In some examples, the mixture also contains additional DNA or RNA primers or additional enzymes such as cutting enzymes, recombinases, helicases, restriction endonucleases, nucleases, or ligases. In some examples, isothermal reactions are permitted for probe-mixture binding at temperatures ranging from 1 minute to 72 hours. In some examples, reactions are permitted for probe-mixture binding at multiple temperature cycles (between 5 and 200 cycles).

[0016] The features and advantages of this invention will be readily understood by those skilled in the art through the description of the examples. Attached Figure Description

[0017] Some specific exemplary examples of the present invention can be understood to some extent by referring to the following description and the accompanying drawings.

[0018] Figure 1 An embodiment of a suitable nucleic acid probe system 10 for use in this invention is provided. The probe system 10 includes a complementary strand C (also referred to herein as a "first strand") and a guard strand P (also referred to herein as a "second strand") designed with respect to a target nucleic acid T (also referred to herein as a "target nucleic acid molecule" or "target nucleic acid strand"). The complementary strand C includes a target-sticky-end complementary region 1 (also referred to herein as a "first region"), a target-homologous complementary region 2 (also referred to herein as a "second region"), and a target-non-homologous complementary region 3 (also referred to herein as a "third region"). The guard strand includes a target-homologous region 4 (also referred to herein as a "fourth region") and a target-non-homologous region 5 (also referred to herein as a "fifth region"). The target includes a target-sticky-end region 6 (also referred to herein as a "sixth region") and a target-validation region 7 (also referred to herein as a "seventh region"). In some embodiments, the target may also include a target upstream region 8, and / or additional unnamed upstream and downstream regions. The target homologous region 4 of the protective strand P may differ in sequence from the target verification region 7 of the target T, for example, in which the protective strand P and the target T are different types of nucleic acids (e.g., RNA versus DNA). As used herein, the term "region" refers to a set of consecutive nucleotide bases that function as a functional unit in hybridization and separation when referring to probe systems or target nucleic acids.

[0019] Figure 2 A provides a typical probe system 10 and its reaction with the target nucleic acid T, and Figure 2 B provides a typical probe system 10 and its reaction with a variant target V that has a single-base difference 12 from target T in the target validation region 7. Now refer to Figure 2 A, Probe system 10 is designed such that probe system 10 interacts with the expected target T(ΔG) o rxn The standard free energy of the hybridization reaction is approximately the same as that of (-Rτln(([P]0-[C]0) / [C]0)) (Equation 2), and ensures a moderate to high yield of the complementary chain C binding to the target T. Now refer to Figure 2 B, Probe system 10 and variant target V with standard free energy ΔG o V The reaction proceeds, ΔG o V Compared to ΔG o rxn large ΔΔG o SNP (that is, ΔG) o V =ΔG o rxn +ΔΔG o SNP), where ΔΔG o SNP This represents the corresponding thermodynamic penalty for a single base change. Compared to the expected target T, this result in probe system 10 shows a very low binding yield with variant target V due to the single base mismatch 12.

[0020] Figure 3 This invention provides various standard free energies of the binding regio components used to calculate the reaction standard free energy (ΔG). o rxn ).

[0021] Figure 4 Provided 50nt non-overlapping sequence data (ΔG) of 46 different BRAF-expressing (exon) mRNAs at different temperatures. o v-TC -ΔG o h-PC The distribution of ΔG values ​​is assumed to be that both the first and second nucleic acid molecules are DNA. As can be seen, the maximum value is often 20 kcal / mol greater than the minimum value. Considering ΔG... o rxn A difference of 1.4 kcal / mol can lead to a 10-fold difference in specificity or sensitivity, and the results here confirm ΔG o v-TC -ΔG o h-PC This should be considered as a design of the probe described in this paper, thereby improving upon existing technical design parameters.

[0022] Figure 5 Different thermodynamic values ​​(ΔG) were provided. o label =ΔG o F -ΔG o FQ The standard free energy contribution of ). In this example, the labeling of the protecting chain P is the quencher Q, which is specific for the fluorophore F of the complementary chain C.

[0023] Figure 6 This is an illustration of one aspect of the method of the present invention for adjusting the behavior of a probe system. Specifically, in addition to adjusting the reaction standard free energy (ΔG) by adding or removing base stacks (adjusting the value of Equation 1), o rxnThis paper proposes a method to control the trade-off between probe specificity and sensitivity by adjusting the stoichiometry (the ratio of P to C concentrations) and provides a more efficient way to do so (adjusting the value of Equation 2). Here, it is assumed that the target concentration is less than the first concentration, and the sensitivity is calculated as the expected equilibrium binding rate of the target [TC] / ([T]+[TC]), and the specificity is calculated as 1 minus the binding rate of the target variant 1-[VC] / ([V]+[VC])=[V] / ([V]+[VC]). In this figure, the difference between the variant and the target is a single base, and ΔΔG at 37°C. o SNP = +2 kcal / mol. ΔG is obtained in the computer simulation through calculation using... o rxn The actual ΔG differs by as much as 5 kcal / mol o rxn To correct the probe system's inability to make ΔG o rxn In cases of precise calculation, adjusting the stoichiometry of the P to C ratio is also beneficial.

[0024] Figure 7 This indicates the design of a variant probe system. Figure 7 A indicates a probe system with opposite 5' / 3' orientation. Figure 7 B indicates a probe system in which region 1 is embedded in region 2, or where region 1 exists between regions 2 and 3. Figure 7 C indicates a probe system in which regions 2 and 4 are not completely complementary, or regions 3 and 5 are not completely complementary.

[0025] Figure 8 Schematic diagrams are provided showing different desired target binding rates and the trade-offs between specificity and sensitivity. The standard reaction free energy ΔG is determined by Equation 1 (or Equation 3 in the case of labeling). o rxn A deviation from the free energy by X (Equation 2 or -Rτln(([P]0-[C]0) / [C]0)) will also alter the target binding yield. For a positive value of X, specificity (for target variant V) will increase, but sensitivity (rate) will decrease. For a negative value of X, sensitivity will increase, but specificity will decrease. For specific applications, specificity and sensitivity are more important and can be finely tuned using the improved thermodynamics described in this invention.

[0026] Figure 9 An exemplary probe of the present invention (Example 1) is provided, which is effective at nucleotides 11-30, τ = 37°C, and [Na+]. + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxnThe calculated value is +0.15 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 0.78 to make X = 0. With ([P]0 - [C]0) / [C]0 = 7.8, X is +1.42 kcal / mol, and with ([P]0 - [C]0) / [C]0 = 0.10, X is -1.27 kcal / mol.

[0027] Figure 10 An exemplary probe of the present invention (Example 2) is provided, which is effective at nucleotides 71-90, τ = 37°C, and [Na+]. + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculation is -0.61 kcal / mol and it is recommended that ([P]0 - [C]0) / [C]0 = 2.69 to make X = 0.

[0028] Figure 11 An exemplary probe of the present invention (Example 3) is provided, which is effective at nucleotides 131-160, τ = 37°C, and [Na+]. + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculation yields -1.54 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 12.14 to make X = 0.

[0029] Figure 12 An exemplary probe of the present invention (Example 4) is provided, which is effective at nucleotides 191-220, τ = 37°C, and [Na] + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculation yields -0.46 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 2.11 to make X = 0.

[0030] Figure 13 An exemplary probe of the present invention (Example 5) is provided, which is effective at nucleotides 251-280, τ = 37°C, and [Na+]. + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculated value is -1.49 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 11.2 to make X = 0.

[0031] Figure 14An exemplary probe of the present invention (Example 6) is provided, which is effective at nucleotides 311-350, τ = 52°C, and [Na] + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculation is -0.22 kcal / mol and it is recommended that ([P]0 - [C]0) / [C]0 = 1.43 to make X = 0.

[0032] Figure 15 An exemplary probe of the present invention (Example 7) is provided, which is effective at nucleotides 431-460, τ = 65°C, and [Na+]. + ]=1M BRAF-targeting mRNA subsequence. Based on literature parameters, ΔG o rxn The calculation is +1.03 kcal / mol and it is recommended that ([P]0 - [C]0) / [C]0 = 0.19 to make X = 0.

[0033] Figure 16 An exemplary probe of the present invention (Example 8) is provided in another direction, which is effective at nucleotides 491-520, τ = 37°C, and [Na] + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculated value is -0.27 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 1.55 to make X = 0.

[0034] Figure 17 An exemplary embodiment of the present invention (Example 9) is provided, which contains an intentional single nucleotide mismatch in the target-homology region (fourth region) of the protective strand, at nucleotides 551-580, τ = 37°C, [Na] + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculated value is -0.37 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 1.82 to make X = 0.

[0035] Figure 18 An exemplary probe of the present invention (Example 10) is provided, which is effective at nucleotides 611-630, τ = 25°C, and [Na] + [] = 1M mRNA subsequence targeting BRAF expression. Based on literature parameters, ΔG o rxn The calculation is -0.66 kcal / mol and it is recommended that ([P]0 - [C]0) / [C]0 = 3.0 to make X = 0.

[0036] Figure 19 An exemplary probe of the present invention (Example 11) is provided, which is effective at nucleotides 671-700, τ = 25°C, and [Na] + [ ] = mRNA subsequence targeting BRAF expression under 1M, 30% formamide. Based on literature parameters and the assumption that 1% formamide corresponds to a temperature increase of 0.6℃, ΔG o rxn The calculated value is 0.32 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 0.58 to make X = 0.

[0037] Figure 20 An exemplary probe of the present invention (Example 12) is provided, which is effective at nucleotides 671-700, τ = 62°C, and [Mg... + Target DNA sequence at 3mM. Based on literature parameters, ΔG o rxn The calculated value is -3.07 kcal / mol, and it is recommended that ([P]0 - [C]0) / [C]0 = 100 to make X = 0.

[0038] Figure 21 A schematic view of hotspot multiplex PCR using the probes of the present invention as primers is provided. A sample 20 containing the desired target nucleic acid molecule 22 is mixed with enzyme 23, forward primer 24, and reverse primer sets 10a-10d. The target nucleic acid molecule 22 comprises a single-base mutation located at a locus adjacent to another locus (“hotspot”) and is typically detected by standard PCR primers. The probes of the present invention have a unique advantage in hotspot multiplex PCR primers because they are specific for single nucleotide mismatches along the full length of the primer. Furthermore, the application of the probes of the present invention as PCR primers (primarily double-stranded) will suppress the formation of primer dimers, which often limit the multiplex amplification capability of PCR. By using different fluorescence channels (1, 2, 3, and 4), each target can be quantified with very few unwanted cross-interactions.

[0039] Figure 22 An exemplary hotspot multiplex PCR reverse primer set is provided, which uses the probes of this invention to target four different NRAS codon 61 mutations. Sequence design and energy calculations are based on the above description of the design, and the [P]0 / [C]0 ratio that theoretically makes X=0 is calculated for each primer system.

[0040] Figure 23Probes targeting DNA targets can be finely tuned by varying the [P]0 / [C]0 ratio. The probes in this diagram are designed to bind to the same DNA target at different standard reaction free energies. Each protecting strand is modified with IowaBlack RQ quencher at 5', and each complementary strand is modified with a TAMRA fluorophore at 3'. Hybridization rates were experimentally determined by fluorescence from different ([P]0-[C]0) / [C]0 ratios. The results indicate that each probe shown in this diagram is tunable in both specificity and sensitivity. All experiments were performed using 1X PBS at 25°C.

[0041] Figure 24 This provides probes for finely tuning the target RNA sequence (synthetic miR-122) by changing the [P]0 / [C]0 ratio. The probe design method, in addition to using RNA-DNA binding parameters, is related to... Figure 22 Similarly, the experimental procedure was the same as for the DNA target. The results showed that the sensitivity / specificity trade-off of the probe against the RNA target is also adjustable.

[0042] Figure 25 A schematic view is provided showing the use of the probes of this invention as self-reporting primers to selectively amplify target nucleic acid molecules. The first nucleic acid strand of primer 10 contains a fluorophore F at one end, and the second nucleic acid strand of primer 10 contains a quencher Q at the other end. During amplification process 30, after hybridization with the desired target nucleic acid molecule 25, the fluorescence signal increases with the diffusion of the second nucleic acid strand of primer P, indicating the formation of amplicon 26.

[0043] Figure 26 A schematic view is provided showing the amplification of target nucleic acid molecules using the probes of this invention as fluorophores to quantify the amount of desired amplicon formed during amplification. The sample may include the desired target nucleic acid molecule 22, which is mixed with enzyme 23, forward primer 27, reverse primer (not shown), and fluorophore-labeled probe 10. The first nucleic acid strand of probe C is functionalized with an internal fluorophore F and a quencher at the 3' end, thus the probe is natively dark due to the close proximity of the fluorophore and quencher. The forward primer and the first nucleic acid strand of probe C hybridize with the desired target nucleotide molecule in an annealing process 31, while the second nucleic acid strand is replaced by the target nucleic acid molecule. In the expansion process 32, enzyme 23, having exonuclease activity, extends the primer and cleaves the phosphodiester bond of the first nucleic acid strand, resulting in an enhancement of the fluorescence signal.

[0044] While the invention is subject to various modifications and alternatives, specific exemplary examples have been shown in the accompanying drawings and these embodiments are described in more detail herein. However, it should be understood that the description of specific exemplary examples is not intended to limit the invention to the specific forms disclosed, but rather this disclosure will cover all modifications and equivalents partially described by the appended claims.

[0045] Invention Description

[0046] The nucleic acid probe system described herein offers several advantages over previously described systems. First, the methods and compositions described herein provide more economical DNA probes for testing RNA targets with specific sequences; since RNA hybridization is generally less specific than DNA hybridization, DNA probes targeting RNA targets also exhibit improved specificity. Furthermore, thanks to robust single-nucleotide specificity, the methods and compositions described herein allow for the use of modified nucleic acid probes, such as those combined with 2'-O-methylnucleotides or locked nucleic acids (LNAs); these modified nucleic acid probes can possess desirable properties such as nuclease resistance. Second, by modifying the relative concentrations of protectants and complements in the probe system, the methods and compositions described herein provide finely tunable specificity and sensitivity performance. Additionally, the probe system has two other desirable features: the probes described herein are highly specific and can operate over a wide temperature and salt concentration range, and are therefore functionally reliable under many different experimental conditions. For example, a single-base change results in approximately a 30-fold difference in binding efficiency between temperatures ranging from 10°C to 70°C. Finally, the probes described herein are kinetically rapid. For example, the probes of this invention interact with target nucleic acid molecules ten times more readily than through hybridization.

[0047] An overview of the reaction between the probe system 10, consistent with the present invention, and its intended target T is shown in Figure 2 A and B. In this example, probe system 10 consists of a protecting oligonucleotide / chain P and a complementary oligonucleotide / chain C, with the protecting chain P present in excess of the complementary chain C. The protecting chain P and the complementary chain C can hybridize to form a partially double-stranded complex; this is true regardless of whether the protecting chain P and the complementary chain C are separately introduced into the target T or pre-reacted to form the complex. Additionally, in some examples, besides the partially double-stranded complex of the protecting chain and the complementary chain, an excess of the complementary chain C or the protecting chain P allows the excess chain to exist as a single-stranded molecule. Figure 2 In A, the reaction between the target T and the probe system 10 is made to have a reaction standard free energy (ΔG) equal to (-Rτln(([P]0-[C]0) / [C]0)]). o rxn The concentration ratio [P]0 / [C]0 is chosen in a manner that allows for selection of the target molecule concentration ratio. This results in, in some cases, half of all target molecules T in the sample at equilibrium binding to the complementary chain C. See also Figure 2 B, Target variant V, which differs from target T in the target-verification 7 or target-sticky end 6 region due to a single base, will have a higher positive standard free energy (ΔG). o V It combines with a significantly lower equilibrium yield (e.g., 2%).

[0048] The protective strand P and the complementary strand C sequences are designed based on the sequence of the intended target T. For example... Figure 1 As shown, each strand is conceptually divided into multiple non-overlapping regions. It is important to note that the target-validation region 7 (also referred to as the "seventh region") and the target-homologous region 4 (also referred to as the "fourth region"), both partially or completely complementary to the target-homologous-complementary region 2 (also referred to herein as the "second region"), can have different sequences. For example, an RNA target may have a target-validation region 7 containing uracil, while the DNA protective strand P may include thymine in the target-homologous region 4. As another example, region 4 may partially mismatch with region 2 at certain sites, while region 7 may perfectly match region 2. As yet another example, both regions 4 and 7 may partially mismatch with region 2, but at different nucleotide bases.

[0049] The standard reaction free energy of an unlabeled probe system is given by ΔG. o rxn =ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC The formula provided is also referenced herein and in the claims as "Formula 1". The standard reaction free energy of a probe system having functionalized groups or labels is given by ΔG. o rxn =ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC )+ΔG o 标签 Provided, which is also referenced herein and in the claims as “Formula 3”. It should be understood that all standard free energy terms used herein are evaluated under the temperature and buffer conditions under which the composition is applied to the target nucleic acid molecule.

[0050] like Figure 3 and 5As shown, Equations 1 and 3 consist of multiple portions representing standard free energies of hybridization between various regions of the protective / complementary / target nucleic acid strands. As indicated above, the term ΔG... o t-TC The standard free energy represents the hybridization between the target-sticky-end region 6 of the target nucleic acid T of probe system 10 and the target-sticky-end complementary region 1 of the complementary strand C. These regions may be partially or completely complementary. In this example, the term "partially complementary" is defined as the nucleotides in the first region being more than 60% complementary to the paired nucleotides in the sixth region. However, it should be understood that the term "partially complementary" may have different meanings in other paired sequences.

[0051] Term ΔG o nh -PC represents the standard free energy of hybridization between the target-non-homologous region 5 of the protective strand P and the target-non-homologous-complementary region 3 of the complementary strand C. In this example, the term "partial complementarity" is defined as the nucleotides in the third region being more than 60% complementary to the aligned nucleotides in the fifth region.

[0052] Term ΔG o v-TC This represents the standard free energy of hybridization between target-validation region 7 of target nucleic acid T and target-homology-complementarity region 2 of complementary strand C. In this example, the term "partial complementarity" is defined as nucleotides in region 2 being more than 60% complementary to the aligned nucleotides in region 7.

[0053] Term ΔG o h -PC represents the standard free energy of hybridization between target-homologous region 4 of the protecting strand P and target-homologous-complementary region 2 of the complementary strand C. These regions can be partially complementary or fully complementary. In this example, the term "partially complementary" is defined as nucleotides in the second region being more than 60% complementary to the aligned nucleotides in the fourth region.

[0054] Term ΔG o label Equal to the standard free energy (ΔG) of the label on the complementary chain o F Subtract the standard free energy of the interaction between the marker and the guard chain, including any other functionalized groups on the guard chain. Figure 5 In the example, the marker (Q) of the guard chain is a quencher specific to the fluorophore (F) of the complementary chain.

[0055] Still referencing Figure 1In some examples, the probe system sequence is designed such that (1) there is almost no secondary structure in the target-sticky-end-complementary region 1, and (2) there is almost no binding between the target-upstream region 8 and the target-non-homologous-complementary region 3. Here, "almost no secondary structure" in the target-sticky-end-complementary region is defined as less than 50% of the nucleotides in the region being in a double-stranded state in the minimum free energy structure being evaluated, calculated under operating temperature and salinity conditions. Here, "almost no binding" between the target-upstream region and the target-non-homologous-complementary region 3 is defined as less than 50% of the nucleotides in the target-non-homologous-complementary region 3 being in a double-stranded state in the minimum free energy structure being evaluated, calculated under operating temperature and salinity conditions.

[0056] In addition to the measured standard free energy of the reaction (ΔG) o rxn For example, according to Formula 1, the probe design of the present invention also includes consideration of the relative concentrations of the guard strand and the complementary strand of the probe. This allows for fine-tuning of the reaction by changing the ratio of the guard strand to the complementary strand, independent of the probe sequence design. Therefore, in one example, the design of the nucleic acid hybridization probe system of the present invention is based on:

[0057] ΔG o rxn =ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC )=-Rτln(([P]0-[C]0) / [C]0)+X

[0058] or

[0059] ΔG o rxn =ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC )+ΔG o label =-Rτln(([P]0-[C]0) / [C]0)+X

[0060] Where X is a value between -5 kcal / mol and +5 kcal / mol. Higher positive X values ​​contribute to higher specificity, and the X value also allows users to control the trade-off between polymer sensitivity and polymer specificity.

[0061] It should be understood that ΔG o The value is calculated only approximately based on currently available literature values, while the probe of the claims is calculated using the actual ΔG. o As described and defined. Based on our ΔG o Experimental studies of the values, based on currently available parameters and software calculations, show that the actual values ​​differ by as much as 3 kcal / mol or 15%, whichever is greater.

[0062] In contrast, WO2012 / 058488 describes the design of nucleic acid hybridization probes, where the main design constraint is ΔG. o t-TC ≈ΔG o nh-PC In the manner described in this invention, "approximately equal to" is defined as a difference of less than 10%. In one embodiment, since the required X value differs significantly from 0, the standard free energy ΔG of the probe of this invention is... o t-TC and ΔG o nh-PC The difference is greater than 10%. In another implementation, due to (ΔG) o v-TC -ΔG o h-PC The difference between 0 and 0 is significant; therefore, the standard free energy ΔG of the probe in this invention is... o t-TC and ΔG o nh-PC The difference is greater than 10%. In another embodiment, since the difference between ([P]0-[C]0) / [C]0 and 1 is significant, the standard free energy ΔG of the probe of the present invention is therefore... o t-TC and ΔG o nh-PC The difference is greater than 10%. In another implementation, due to ΔG o label The difference from 0 is significant, therefore the standard free energy ΔG of the probe of this invention is... o t-TC and ΔG o nh-PC The difference is greater than 10%.

[0063] Therefore, the probe system of the present invention is in ΔG o v-TC ΔG o h-PC ΔG o label X and The considerations differ from existing technologies. In many nucleotide sequences, regions 4 and 7 are set differently, regarding the term ΔG. o v-TC ΔG o h-PC Negligence can lead to poor probe design, particularly when using fluorophores or other labels, and misinterpretation of the term ΔG. o label Negligence in this area can lead to poor probe design; neglect of X can hinder different trade-offs between specificity and sensitivity; and neglect of stoichiometry can impede fine-tuning of probe system behavior independent of sequence design. Furthermore, it can cause probes to perform poorly at certain P and C stoichiometry ratios. These will be discussed in more detail below.

[0064] First, return to the reference. Figure 2 A. The target-validation region 7 of target T and the target-homology region 4 of the protective strand P differ in sequence and thermodynamic properties for a variety of reasons, which is important in cases where T and P are different nucleic acid types. For example, target T may be an RNA molecule for scientific / clinical interest, while protective strand P may be a DNA molecule for economic / synthetic efficiency. As another example, protective strand P may include modified nucleic acids such as 2'-O-methylnucleotides or locked nucleic acids (LNAs). As yet another example, regions 4 and 7 may both be DNA, but differ in nucleotide sequence to benefit from enhanced kinetics or a reduced, undesirable biological response.

[0065] When the target-verification region 7 of target T and the target-homogeneous region 4 of the guard chain P are different, ΔG o v-TC and ΔG o h-PC They are not equal, and ΔG must be considered during the design process of the driving probe system. o rxn This should be taken into consideration. ΔG o v-TC -ΔG o h-PC The value can deviate significantly from 0. Now refer to... Figure 4 The distribution of these values ​​for non-overlapping subsequences (each 50 nt long) of 46 different BRAF transcript RNAs compared to homologous DNA sequences bound to DNA complements is shown, using RNA-DNA hybridization thermodynamic values ​​obtained from Sugimoto et al. [7] and DNA-DNA hybridization thermodynamic values ​​obtained from Santa Lucia and Hicks [8]. As can be seen, not only ΔG o v-TC -ΔG o h-PCThe value ranges from -20 kcal / mol to +20 kcal / mol, and this value also depends on temperature. In comparison, even ΔG... o rxn Even a difference of 1 kcal / mol can lead to significant changes in sensitivity and / or specificity.

[0066] The detection of RNA target T using DNA probes (P and C) only requires consideration of ΔG. o v-TC -ΔG o h-PC One application. Because T and P are different types of nucleic acids (RNA, DNA, LNA, PNA, phosphate-thioester DNA, 2'-methoxynucleotides, etc.) or due to minor changes in the sequence, there are other variations in the probe system where the target-homological region of the protective strand P differs from the target-validation region of the target T, which will be discussed in detail below.

[0067] By ignoring ΔG o v-TC -ΔG o h-PC It must be assumed that the total value of this item is 0 kcal / mol. This assumption applies only if the target-homologous region 4 of the protective strand P and the target-validation region 7 of the target T have the same characteristics and sequence, for example, if the application of a DNA target using a DNA protective strand has the same nucleotide sequence as regions 7 and 4.

[0068] Second, many applications of nucleic acid detection or imaging use labeling to aid in the visualization of the presence or quantification of target nucleic acids. These labels can be organic fluorophores, metal nanoparticles, or haptens that recruit antibodies. Typically, these labels can possess significant thermodynamic effects, stabilizing or destabilizing nucleic acid hybridization. Appropriate design of labeled probe systems should take into account... Figure 5 The difference between the marker and the standard free energy of the protective chain and the target is shown in the figure.

[0069] Third, as mentioned above, the relative concentrations of the protecting strand P and the complementary compound C play a crucial role in regulating the probe system of this invention, and this parameter exists independently of the sequence design of the probe system. Given the current imperfect understanding of DNA and RNA hybridization thermodynamics and labeling thermodynamics, the ability to regulate the performance of specific designed and synthesized probe systems is essential for the practical applications involving these probe systems.

[0070] To understand the role of the relative concentrations of P and C in regulating the performance of a probe system, the equilibrium of the reaction between the target and the probe system must be considered. The entire chemical reaction can be described as follows.

[0071]

[0072] Typically, the concentration of the target (biological DNA or RNA molecule) is much lower than that of the probe components P and PC; higher concentrations of P and PC help drive the reaction to reach equilibrium quickly. A useful indicator for judging the behavior of the reaction is the yield or sensitivity of the probe system to the target T, which can be expressed as... When the sensitivity is approximately 50%, i.e., when the equilibrium concentration of unbound T equals the equilibrium concentration of T bound to C ([T] = [TC]), the fold difference in the variant target V ([TC] / [VC]) is preferably within 2. The value of the equilibrium constant Keq can be obtained by analyzing [T] = [TC] using the following formula.

[0073]

[0074] The standard free energy of a reaction can be related to the reaction equilibrium constant by the following formula.

[0075]

[0076] In the above equations, [P]0 represents the initial concentration of the guarding chain and [C]0 represents the initial concentration of the complement. Since the target concentration [T] is usually lower than the concentration of either the guarding chain or the probe, the equilibrium concentrations of [P] and [PC] can be approximated as [P]0 - [C]0 and [C]0, respectively. Since the scale remains constant, the concentrations used for [P]0 and [C]0 can be the high concentration of the raw material added to the sample, or the final concentration obtained by diluting the sample. It is important to note that [P]0 and [C]0 refer to the total concentration of P and C, including those present in some double-stranded PC species. Another way to express this formula is ([P]0) = ... free ]0 / [PC]0), where [P free [0] represents the initial concentration of free P and [PC]0 represents the initial concentration of PC.

[0077] For use with the probe system of the present invention, the concentration of [P]0 can be lower, the same as, or higher than, but generally higher than, the concentration of [C]0. For example, the concentration of [P]0 can be about 1.01 times to about 10,000 times, about 1.1 times to about 1,000 times, or about 1.2 times to about 100 times, and includes any intermediate range between any of the above-mentioned ranges.

[0078] In one example, the behavior of the probe can be tuned by designing the probe system to achieve approximately 50% sensitivity, such that ΔG o rxn Approximately 0, or from about -5 kcal / mol to about +5 kcal / mol, then adjust [P]0 and [C]0 to meet the requirements. Importantly, due to the adjustment of ΔG by adding or removing base pairs / piles o rxn The resulting coarse-grained properties make it virtually impossible to achieve 50% sensitivity (or any other desired sensitivity) without adjusting the probe system via [P]0 and [C]0.

[0079] Figure 6 This indicates that at 37℃ and 1M Na + The next single additional base pair makes ΔG o rxn The value varies between -0.6 kcal / mol and -2.2 kcal / mol.

[0080] Therefore, this invention provides a method for finely adjusting ΔG by adjusting the stoichiometry of the protection chain P and the complementary compound C. o rxn A new concept. The accuracy of the stoichiometric ratio of P and C is limited only by the accuracy of the liquid handling system (e.g., pipette accuracy) and is typically controllable within 2%. This 2% stoichiometric accuracy, in turn, leads to the adjustment of probe performance – Rτln(1.02) = -0.012 kcal / mol and ΔG o rxn The resolution accuracy is the same. Therefore, adjusting the thermodynamic ratio by the stoichiometry of P and C is more than 50 times finer than the existing method of adjusting thermodynamics by adding base pairs (-0.012 kcal / mol vs. -0.60 kcal / mol). The adjustment of the stoichiometry of P and C can be achieved during the design phase or dynamically optimized iteratively as a probe for a specific application.

[0081] Figure 22 and Figure 23 The experimental results provided show that adjusting the ratio is necessary to adjust the specificity / sensitivity tradeoff. The effectiveness. Figure 22 The sequence designs of four different probes directed to a DNA target are shown (each designed with a different ΔG). o rxn ), and the observed yield of the DNA target for each probe at different ([P]0-[C]0) / [C]0 values. Figure 23 The sequence designs of five different probes targeting RNA targets are described (each designed to be ΔG). o rxn The observed yield of the RNA target for each probe varies depending on the ([P]0-[C]0) / [C]0 value. As previously taught, a larger ([P]0-[C]0) / [C]0 results in a monotonically decreasing hybridization yield between the target and the probe.

[0082] In another aspect, the present invention provides a probe system wherein the following is not satisfied: Instead, it provides subtle variations, where the values ​​are not equal, to achieve different trade-offs between specificity and sensitivity. Therefore, the thermodynamic properties of the probe system of the present invention can be described as follows:

[0083]

[0084] Where X deviates from 0. In one example, the value of X ranges from approximately -5 kcal / mol to approximately +5 kcal / mol. For positive X values, specificity (against target variant V) will increase, but sensitivity (yield) will decrease. For negative X values, such as... Figure 8 The sensitivity shown will increase, but the specificity will decrease. In practice, some applications (such as those involving rare alleles) may require higher specificity at a lower cost than higher sensitivity, and vice versa. The finely tuned thermodynamics method of the present invention is particularly useful for these applications requiring complex tuning of both sensitivity and specificity (see also the variants).

[0085] In another aspect, the present invention provides minute sequence differences between the target-verification and target-homologous regions. Both the target-verification region (of target T) and the target-homologous region (of guard strand P) are intended to be complementary to the target-homologous-complementary region (of complement C). However, there may be cases where it is desirable to have minute sequence modifications in the target-verification and / or target-homologous regions such that the target-verification and / or target-homologous regions are only partially complementary to the target-homologous-complementary region. For this purpose, in examples where more than 60% of the bases in the target-homologous-complementary region are complementary to the target-verification region, and in examples where more than 60% of the bases in the target-homologous-complementary region are complementary to the target-homologous region, the resulting probes are consistent with the principles of the probe structures described herein.

[0086] In addition, the present invention provides a probe system, such as Figure 7 As shown, the 5' to 3' orientation of the protective strand and complement is opposite to the position of the non-homologous and sticky end regions. Modern nucleic acid synthesis proceeds from the 3' end to the 5' end, resulting in truncation and deletion concentrated at the 5' end. Therefore, it is expected that... Figure 1-6 The original orientation shown is ideal because the cutoffs on the guard chain and complement will tend to positively balance each other, maintaining the required ΔG. o rxn On the contrary, in Figure 7 In the design approach shown, the cutoffs in both the guard chain and the complement tend to reduce ΔG. o rxnThis negative effect reduces the reliability and specificity of the probe system. These effects are mitigated when the protecting strand and complementary oligonucleotides are purified post-synthesized by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis (PAGE).

[0087] At the standard free energy of the reaction (ΔG) o rxn In the analysis, the ΔG of unstructured oligonucleotide formation... o The standard free energy is defined as 0. The equilibrium constant (Kt) of the reaction between the target T and the probe system (P and C) is... eq The standard free energy ΔG of the reaction can be directly obtained from the following formula. o rxn Work out:

[0088]

[0089] Where R = 8.314 J / mol, K is the ideal gas constant (or Boltzmann constant), and τ is the ambient temperature at the Kelvin temperature.

[0090] In the design of the probe system of this invention, the reaction ΔG o rxn Decomposed into multiple ΔG o The sum, ΔG o This represents the standard hybridization free energy (e.g., ΔG) of each region from the complementary strand to the target strand and from the complementary strand to the guard strand. o nh-PC This represents hybridization from a target-non-homologous region to a target-homologous complementary region. The values ​​of these terms can be roughly calculated by adding the standard free energy of the base stack as detailed below, although the standard free energy values ​​provided in the current literature are incomplete and have limited precision. Experimental testing is needed to determine the true ΔG for each probe. o rxn Values ​​are provided, but literature guidelines offer rough estimates (typically within 3 kcal / mol or 15%) of ΔG. o rxn The estimate.

[0091] In one example, the standard free energy of hybridization between regions of the probe system of the present invention is calculated based on a base pair stacking method. In this method, two adjacent base pairs comprise a stack having a specified enthalpy (ΔH). o ) and entropy (ΔS) o The standard free energy (ΔG) of each pile at a specific temperature τ (Kelvin temperature). o It can be derived from the equation ΔG o =ΔH o -τΔS oCalculation. The standard free energies of several stacks can be summed to evaluate the standard free energy of the bonded region. For example, the standard free energy of the 'CTC' region paired with the 'GAG' region is the sum of the standard free energy of stack 'CT / GA' and stack 'TC / AG'. At 37°C and 1M Na... + The standard free energy of the 'CT / GA' region is -1.28 kcal / mol and the standard free energy of the 'TC / AG' region is -1.30 kcal / mol. Therefore, the standard free energy of the 'CTC' region, which is paired with the 'GAG' region, is -2.58 kcal / mol.

[0092]

[0093] ΔH of DNA-DNA stacks o and ΔS o The values ​​are based on the study published by SantaLucia and Hicks, and are shown in Table 1. The standard enthalpy change and standard entropy change of the RNA-DNA stack are based on the study published by Sugimoto et al., and are shown in Table 2. The standard enthalpy change and standard entropy change of the RNA-RNA stack are based on the study published by Turner et al., and are shown in Table 3. ΔH of the base stack o The values ​​are all accepted by the literature without considering salinity. In contrast, the ΔS of the base stack... o By 0.368*ln([Na + The cal / mol*K ratio is adjusted because the electrostatic shielding properties of cations disregard the similarity of nucleotide bases. Additionally, divalent cations (such as Mg²⁺) are also considered. 2+ Formamide can also be used in reaction solutions; the effects of divalent cations on base pairing thermodynamics are described in the literature (e.g., Owczarzy, *Biochemistry*, 2008). Finally, denaturing agents such as formamide can be used to promote hybridization reactions, especially in in situ hybridization applications. According to the literature, for nucleic acid base pairing thermodynamics purposes, each percentage point of water effectively replaced by formamide increased the temperature by 0.6 °C; see Blake and Delcoult, *Nucleic Acids Research*, 1996.

[0094] Table 1.1 Thermodynamic parameters of DNA Watson-Crick pairing in 1M NaCl.

[0095]

[0096] Table 2.1 Thermodynamic parameters of RNA-DNA duplex pairing in M ​​NaCl.

[0097]

[0098] Table 3.1 Thermodynamic parameters of RNA-RNA duplex pairing in M ​​NaCl.

[0099]

[0100] In one example, the standard free energy of hybridization reaction in each region of the probe system of the present invention (ΔG from Equation 1 or 3) o rxn The calculation is as follows.

[0101] ΔG o t-TC (Hybridization of the target-sticky-end region (region 6) to the target-sticky-end complementary region (region 1)) consists of all the sticky-end region nucleic acid stacks, adjacent stacks, and the initial energy loss (ΔG). o ini The standard free energy (due to the entropy loss caused by the orientation of the two nucleic acid molecules for hybridization) is added together. ΔG o ini The value can be obtained through ΔG o =ΔH o -τΔS o From ΔH o ini and ΔS o ini calculate

[0102]

[0103] ΔG o t-TC =ΔG o 邻近 +ΔG o ini +∑ΔG o t-堆

[0104] For the DNA-DNA hybridization provided in Table 1, ΔH o ini =0.2kcal / mol and ΔS o ini = -5.7 cal / (mol·K). For RNA-DNA hybridization provided in Table 2, ΔH o ini =1.9 kcal / mol and ΔS o ini = -3.9 cal / (mol·K). For RNA-RNA hybridization provided in Table 3, ΔH o ini =0.0 kcal / mol and ΔS oini = -10.8 cal / (mol·K).

[0105] In one example, the probe described in this invention has a ΔG value of about -2 kcal / mol to about -16 kcal / mol, about -5 kcal / mol to about -13 kcal / mol, and about -7 kcal / mol to about -10 kcal / mol under operating conditions. o t-TC .

[0106] ΔG o nh-PC (The hybridization of the target of the protective chain P to the non-homologous region 5 to the target of the complementary compound C to the non-homologous-complementary region 3) is determined by all the piles in the non-homologous region, the adjacent piles in the homologous region, and the hybridization initiation energy ΔG. o ini The standard free energies are summed. The standard free energy of each heap and the initial standard free energy are calculated based on the methods discussed above.

[0107] ΔG o v-TC (The hybridization of target-validation region 7 of target T to target-homologous-complementary region 2 of complement C) equals the sum of all nucleic acid stacks in the target-validation region. Each standard free energy is calculated based on the method discussed above. In this example, the initial energy ΔG o ini It was not used in the calculation for this item.

[0108] ΔG o h-PC (The hybridization of target-homologous region 4 of the protective strand P to target-homologous-complementary region 2 of the complement C) is equal to the sum of all nucleic acid stacks in the target-homologous region. Each standard free energy is calculated based on the method discussed above. In this example, the initial energy ΔG o ini It was not used in the calculation for this item.

[0109] In one example, the sum of the standard free energies (ΔG) of hybridization between the target-sticky-end-complementary region (Region 1) and the target-sticky-end region (Region 6) and between the target-homogeneous-complementary region (Region 2) and the target-validation region (Region 7) is... o t-TC +ΔG o v-TCMore negative than -7 kcal / mol, for example, from approximately -7 kcal / mol to approximately -70 kcal / mol, from approximately -7 kcal / mol to approximately -50 kcal / mol, and from -7 kcal / mol to approximately -30 kcal / mol. In this example or others, the sum of the standard free energies (ΔG) of hybridization between the target-non-homologous-complementary region (Region 3) and the target-non-homologous region (Region 5) and between the target-homologous region (Region 4) and the target-homologous-complementary region (Region 2) is... o nh-PC +ΔG o h-PC More negative than -10 kcal / mol, for example, from about -10 kcal / mol to about -70 kcal / mol, from about -10 kcal / mol to about -50 kcal / mol, and from -10 kcal / mol to about -30 kcal / mol.

[0110] Besides enzyme-free nucleic acid detection systems, the probes of this invention are useful as primers in PCR applications or other isothermal amplification systems, such as in hotspot multiplex PCR reactions. When using the probes in PCR reactions, unwanted amplification can be minimized with careful design and fine-tuning. Therefore, two or more primer systems targeting different targets can be combined for hotspot multiplex PCR. A schematic diagram of hotspot multiplex PCR is shown in [illustration missing]. Figure 21 Due to the high specificity of primer systems, the target sequences in a multiplex set can be highly similar. The design method for each primer system in the primer set is the same as that for the probes described above. The specificity and sensitivity of each primer system can be adjusted based on experimental results. An example of a primer system for hotspot multiplex PCR is shown below. Figure 22 .

[0111] In one implementation, signal generation methods for PCR or other isothermal amplification systems utilize a fluorophore-modified complement and a quencher-modified guard strand. The guard strand is separated from the complement as amplified, thus the fluorescence signal is proportional to the copy number of the amplified target. Different targets can be quantified simultaneously using fluorophores with non-overlapping spectra. Figure 25 A similar signal generation method using fluorophore-modified complementary compounds and quencher-modified guarding strands as self-reporting primers is shown. Figure 26This paper presents an alternative signal generation method similar to conventional TaqMan probes, using fluorophore- and quencher-modified complementary and unmodified protective chains as probes. In various settings, probes carrying different fluorophores can be used for different desired targets. Unlike conventional TaqMan probes, which can only be used in situations where the desired target height varies, this invention ensures that each TaqMan probe specifically binds to only one target without interfering with the response of other targets.

[0112] TaqMan probes have a unique advantage in distinguishing similar targets.

[0113] Each probe system described in this invention may be composed of DNA, RNA, or analogues thereof, and / or combinations thereof. In some examples, the probe system includes one or more non-natural nucleotides. Integration of non-natural nucleotides into the primers can further improve the performance of the probe system, such as by providing improved per-base binding affinity and enhanced nuclease resistance.

[0114] The probe system described in this invention can also be used in environments that initiate enzymatic reactions; in such environments, the probe system is introduced as a primer system, but the composition and method of action are the same. The primer system described in the specification has high specificity and fine-tuning capabilities, which are advantageous in enzymatic assays of nucleic acids.

[0115] In some instances, the primers described in this invention function as initiating points for polymerase extension, including but not limited to polymerase chain reactions for replicating DNA templates, transcription of RNA products from DNA templates, reverse transcription of DNA products from RNA templates, and isothermal DNA and RNA amplification methods such as nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), isothermal exponential amplification reaction (EXPAR), cleavage enzyme amplification reaction (NEAR), rolling circle amplification (RCA), and transcription-mediated amplification (TMA). The high specificity of the primers described in this invention makes them suitable for research and clinical applications, in which nucleic acids and subsets of specific sequences are extended and amplified.

[0116] The “target” for the probe system described in this invention can be any single-stranded nucleic acid, such as single-stranded DNA and single-stranded RNA, including single-stranded DNA and RNA obtained by heat shock, asymmetric amplification, competitive binding, and other standard methods in the art. The “target” for the primer system can be any single-stranded (ss) or double-stranded (ds) nucleic acid, such as DNA, RNA, or DNA products of reverse transcription of RNA. In some examples, the target can be a mixture of DNA and RNA (chimera). In other examples, the target includes artificial nucleic acid analogs, such as peptide nucleic acids (Nielsen et al., Science, 254(5037): 1497-500(1991)) or locked nucleic acids (Alexei et al., Tetrahedron, 54(14): 3607-30(1998)). In some examples, the target can be naturally generated (e.g., genomic DNA) or can be synthesized (e.g., from a genomic library). The “naturally occurring” nucleic acid sequence used in this article refers to a sequence present in the nucleic acid molecules of organisms or viruses that exist in nature without human intervention. In some cases, the target is genomic DNA, messenger RNA, ribosomal RNA, microRNA, pre-microRNA, promicroRNA, long non-coding RNA, small RNA, epigenetically modified DNA, epigenetically modified RNA, viral DNA, viral RNA, or piwi-RNA. In some cases, the target nucleic acid is a naturally occurring nucleic acid in an organism or virus. In some cases, the target nucleic acid is the nucleic acid of a pathogenic organism or virus. In some cases, the presence or absence of the target nucleic acid in a subject indicates that the subject has a disease or disorder, or is susceptible to a disease or disorder. In some cases, the presence or absence of the target nucleic acid in a subject indicates a good or poor response to treatments such as drugs used to treat a disease or disorder. In some cases, the presence or absence of the target nucleic acid in a subject indicates that the subject has previously received cancer treatment and is in remission with a risk of relapse.

[0117] The terms “polynucleotide,” “nucleic acid,” “oligonucleotide,” and “nucleic acid molecule” are used interchangeably. They refer to any form of nucleotide polymer, whether deoxyribonucleotides, ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene segments, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched-chain polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure may be conferred before or after polymer assembly. Polynucleotides may be further modified, such as by binding to labeled components. The term “recombinant” polynucleotide means a genomic, cDNA, semi-synthetic, or synthetically derived polynucleotide that is not naturally produced or is linked to another polynucleotide in an unnatural arrangement. The term “isolated nucleic acid” means a polynucleotide or some combination thereof of natural or synthetic origin that (1) is not associated with the cell to which the “isolated nucleic acid” is found in nature, and / or (2) is operatively linked to a polynucleotide to which it is not linked in nature.

[0118] Nucleic acids may also include single- and double-stranded DNA and RNA, as well as any and all forms of nucleic acids comprising modified bases, sugars, and a backbone. The term "nucleic acid" is therefore understood to include, but is not limited to, single- or double-stranded DNA or RNA (which may be in partially single-stranded or partially double-stranded form), cDNA, aptamers, peptide nucleic acids ("PNAs"), 2'-5' DNA (synthetic material with a shortened backbone and base spacing matching the A conformation of DNA; 2'-5' DNA typically does not hybridize with type B DNA but readily hybridizes with RNA), and locked nucleic acids ("LNAs"). Nucleic acid analogs include analogs of known natural nucleotides that have similar or improved binding, base-pairing hybridization properties. "Analogous" forms of purines and pyrimidines are well known in the art and include, but are not limited to, aziridine cytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudorazine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methyl Cytosine, N.sup.6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylpiperidine, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-oxyacetate, pseudouracil, piperidine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate, and 2,6-diaminopurine.The DNA backbone analogs described herein include phosphodiester, thiophosphate, dithiophosphate, methyl, phosphate, alkyl phosphate triester, aminosulfonic acid, 3'-thioacetal, methylene (methylimino), 3'-N-carbamate, morpholinocarbamate, and peptide nucleic acids (PNAs), linked with methylphosphonate or alternating links of methylphosphonate and phosphodiester (Strauss-Soukup, 1997, Biochemistry, 36:8692-8698), and linked with benzylphosphonate, as discussed in U.S. Patent 6,664,057; see also "Oligonucleotides and Analogies, A Practical Approach," edited by F. Eckstein, Oxford University Press, IRL Press (1991); "Antisense Strategies," Annals of the New York Academy of Sciences. *Journal of the American Academy of Sciences*, Vol. 600, edited by Baserga and Denhardt (NYAS 1992); Milligan, 1993, J. Med. Chem. 36: 1923-1937; *Antisense Research and Applications* (1993, CRC Press). The nucleic acids described herein may be extracted from cells or synthesized using any means known to those skilled in the art; for example, nucleic acids may be chemically synthesized or transcribed or reverse-transcribed from cDNA or mRNA.

[0119] The target nucleic acid used herein can be any nucleic acid, such as human nucleic acid, bacterial nucleic acid, or viral nucleic acid. The target nucleic acid sample or a sample containing the target nucleic acid can be, for example, a nucleic acid sample derived from one or more biological samples, including but not limited to whole blood, nucleic acid extracted from whole blood, plasma, nucleic acid extracted from plasma, saliva, feces, urine, buccal or nasal swabs, cells, tissues, or body fluids. Target biological samples can be derived from any source, including but not limited to eukaryotes, plants, animals, vertebrates, fish, mammals, humans, non-humans, bacteria, microorganisms, viruses, biological sources, serum, plasma, blood, urine, semen, lymph, cerebrospinal fluid, amniotic fluid, biopsy, needle aspiration biopsy, cancer, tumors, tissues, cells, cell lysates, crude cell lysates, tissue lysates, tissue culture cells, buccal swabs, mouthwash, feces, mummified tissue, forensic sources, autopsies, archaeological sources, infections, nosocomial infections, production sources, pharmaceutical preparations, biomolecular products, protein preparations, lipid preparations, carbohydrate preparations, inanimate objects, air, soil, sap, metals, fossils, excavated materials, and / or other terrestrial or extraterrestrial materials and sources. Samples can also contain mixtures of materials from one or different sources. For example, the nucleic acids of infected bacteria or viruses can be amplified along with human nucleic acids when nucleic acids from such infected cells or tissues are amplified using the methods of this invention. Useful target sample types include eukaryotic samples, plant samples, animal samples, vertebrate samples, fish samples, mammalian samples, human samples, non-human samples, bacterial samples, microbial samples, viral samples, biological samples, serum samples, plasma samples, blood samples, urine samples, semen samples, lymph samples, cerebrospinal fluid samples, amniotic fluid samples, biopsy samples, needle aspiration biopsy samples, cancer samples, tumor samples, tissue samples, cell samples, cell lysate samples, crude cell lysate samples, tissue lysate samples, tissue culture cell samples, buccal swab samples, mouthwash samples, fecal samples, mummy tissue samples, autopsy samples, archaeological samples, infection samples, nosocomial infection samples, production samples, pharmaceutical preparation samples, biomolecular product samples, protein preparation samples, lipid preparation samples, carbohydrate preparation samples, inanimate object samples, air samples, soil samples, tree sap samples, metal samples, fossil samples, excavated material samples, and / or other terrestrial or extraterrestrial samples. In some examples, the target nucleic acids used herein include repetitive sequences, secondary structures, and / or high G / C content.

[0120] In some examples, the target nucleic acid molecule of interest is approximately 19 to approximately 1,000,000 nucleotides (nt) long. In some examples, the target length is approximately 19 to approximately 100, approximately 100 to approximately 1,000, approximately 1,000 to approximately 10,000, approximately 10,000 to approximately 100,000, or approximately 100,000 to approximately 1,000,000. In some examples, the target length is approximately 20, approximately 100, approximately 200, approximately 300, approximately 400, approximately 500, approximately 600, approximately 700, approximately 800, approximately 900, approximately 1,000, approximately 2,000, approximately 3,000, approximately 4,000, approximately 5,000, approximately 6,000, approximately 7,000, approximately 8,000, approximately 9,000, approximately 10,000, approximately 20,000, approximately 30... 000, approximately 40000, approximately 50000, approximately 60000, approximately 70000, approximately 80000, approximately 90000, approximately 100000, approximately 200000, approximately 300000, approximately 400000, approximately 500000, approximately 600000, approximately 700000, approximately 800000, approximately 900000, or approximately 1,000000 nucleotides. It is understood that the target nucleic acid can be provided in a longer nucleic acid context (e.g., coding sequences or genes in chromosomes or chromosomal segments).

[0121] In some cases, the target of interest is linear, while in others, the target is circular (e.g., plasmid DNA, mitochondrial DNA, or plasmid DNA).

[0122] In some examples, a primer-target system is provided herein. A primer-target system includes one or more nucleic acid targets, a polymerase, and one or more primers (e.g., primer duplexes). The term "primer" includes any of the primers or primer systems described herein. In some examples, the primer-target system described herein includes multiple different primers. In some examples, a primer-target system may include at least two primers that can be used to recognize and, for example, amplify a target nucleic acid molecule. The target nucleic acid molecule can be present, for example, in a single copy or a low copy number, in multiple non-target nucleic acid molecules. Any primer-target system described herein may include conditions similar to those for nucleic acid amplification or sequencing reactions (e.g., similar reagents, reaction temperatures, etc.).

[0123] The kit provided by the present invention comprises (1) at least one complementary strand having a target-homologous-complementary region (region 2), a target-non-homologous-complementary region (region 3), and a target-sticky end-complementary region (region 1), and (2) at least one guarding strand having a target-homologous region (region 4) and a target-non-homologous region (region 5). The kit provided by the present invention comprising at least one primer duplex comprises (1) at least one complementary strand having a target-homologous-complementary region, a target-non-homologous-complementary region, and a target-sticky end-complementary region, and (2) at least one guarding strand having a target-homologous region and a target-non-homologous region.

[0124] Any of the kits described herein may further include a polymerase, including reverse transcriptase. Any of the kits described herein may further include one or more reagents selected from buffers (e.g., KCl, MgCl2, Tris-HCl), dNTPs (e.g., dATP, dCTP, dGTP, dTTP), and water. Any of the kits described herein may include a protecting strand, which is a molar excess of primer. Any of the kits provided herein may further include instructions or a guide for obtaining instructions (e.g., from a website) for using the kit components. Any of the kits provided herein may further include at least one reaction tube, well, lumen, etc.

[0125] Unless otherwise stated, all numerical values ​​used in this specification and claims to indicate the content of each component, properties such as molecular weight, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless stated to the contrary, the numerical parameters described in this specification and appended claims are approximate values ​​and may vary according to the desired properties sought by the invention. In no way is the application of the doctrine of equivalents limited to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures recorded and taking into account the application of commonly used rounding rules.

[0126] The term “a” or “an” when used in conjunction with “comprising” in the claims and specification may mean “a”, but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more.” As used herein, “another” may refer to at least a second or more.

[0127] Any example discussed in this specification can be implemented using any of the methods or compositions of this invention, and vice versa. Furthermore, the compositions of this invention can be used to implement the methods of this invention.

[0128] Throughout this application, the term “about” is used to refer to a value that includes the apparatus, the method for measuring the value, or the inherent variation in the value, including inherent error, caused by differences in the subjects under study.

[0129] The use of the term "or" in the claims means "and / or" unless it is expressly stated that it is only for substitutes or that the substitutes are mutually exclusive.

[0130] As used in this specification and claims, the words “comprising” (and any form of inclusion, such as “containing” and “including”), “having” (and any form of “having”, such as “having” and “owning”), “including” (and any form of “including”, such as “encompassing” and “included”), or “containing” (and any form of “containing”, such as “included” and “included”) are inclusive or open-ended and do not exclude additional unmentioned elements or method steps.

[0131] To facilitate a better understanding of the present invention, some specific embodiments are provided. These embodiments should not be construed as limiting or restricting the full scope of the invention. Example

[0132] Figure 9-20 Twelve examples of DNA probe systems and RNA targets are shown.

[0133] The following examples illustrate the design principles and clarify the standard free energy (ΔG) of the reaction in different regions. o The mathematical operations performed are described, and typical probe systems produced by the methods described in this invention are illustrated. These representative examples cover a range of different biological target sequences and were calculated at many different operating temperatures and salinities. Furthermore, Example 11 illustrates the design of a probe intended to operate at a denatured formamide concentration. The stoichiometric ratio [P]0 / [C]0 required to satisfy the standard free energy values ​​of Equation 1 equal to Equation 2 is also given.

[0134] Table 4: Standard free energy and stoichiometric data of the probes in Examples 1-12.

[0135]

[0136] Example 1

[0137] Example 1 provides a probe targeting the nucleic acid BRAF 11-30, such as Figure 9 As shown. 37℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG)o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that provide values ​​equivalent to those provided in Equation 2 are also provided in Table 4. Finally, the X values ​​provide variations of Equation 2 to obtain values ​​equivalent to those in Equation 1 for the given corresponding stoichiometric ratios, and are 0.00, 1.42, and -1.27 kcal / mol, respectively.

[0138] Example 2

[0139] Example 2 provides a probe targeting the nucleic acid BRAF 71-90, such as Figure 10 As shown. 37℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG ov-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0140] Example 3

[0141] Example 3 provides a probe targeting the nucleic acid BRAF 131-160, such as Figure 11 As shown. 37℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0142] Example 4

[0143] Example 4 provides a probe targeting the nucleic acid BRAF 191-220, such as Figure 12 As shown. 52℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔGo h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0144] Example 5

[0145] Example 5 provides a probe targeting the nucleic acid BRAF 251-280, such as Figure 13 As shown. 65℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔGo v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0146] Example 6

[0147] Example 6 provides a probe targeting the nucleic acid BRAF 311-350, such as Figure 14 As shown. 52℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0148] Example 7

[0149] Example 7 provides a probe targeting the nucleic acid BRAF 431-460, such as Figure 15 As shown. 65℃, 1M Na + The following ΔG of the probe hybridization to the target oValues ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0150] Example 8

[0151] Example 8 provides a probe targeting the nucleic acid BRAF 491-520, such as Figure 16 As shown. 37℃, 1M Na + The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o v-TC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG ot-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0152] Example 9

[0153] Example 9 provides a probe targeting the nucleic acid BRAF 551-580, such as... Figure 17 As shown. 37℃, 1M Na + ΔG of time probe hybridization to target o The following values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG) o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0154] Example 10

[0155] Example 10 provides a probe targeting the nucleic acid BRAF 611-630, such as Figure 18 As shown. 25℃, 1M Na + The following ΔG of the probe hybridization to the targeto Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0156] Example 11

[0157] Example 11 provides a probe targeting the nucleic acid BRAF 670-700, such as... Figure 19 As shown. 25℃, 1M Na + The following ΔG values ​​were observed when the probe hybridized to the target in 30% formamide. o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG) o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔGo t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0158] Example 12 provides a probe targeting a DNA nucleic acid, such as... Figure 20 As shown. 62℃, 3mM Mg 2+ The following ΔG of the probe hybridization to the target o Values ​​are provided in Table 4: (1) Hybridization of target homologous complementary region 2 of complementary strand C to target homologous region 4 of protective strand P (ΔG o h-PC (2) Hybridization of the target-non-homologous-complementary region 3 of complementary strand C to the target-non-homologous region 5 of protective strand P (ΔG) o nh-PC (3) Hybridization of target-homologous complementary region 2 of complementary strand C to target-verification region 7 of target T (ΔG) o v-TC (4) Hybridization of the target-sticky end-complementary region 1 of complementary strand C to the target-sticky end region 6 of target T (ΔG) o t-TC ); and (5)ΔG o rxn For ΔG o t-TC -ΔG o nh-PC +(ΔG o v-TC -ΔG o h-PC (Equation 1). Furthermore, let ΔG according to Equation 1... o rxn The stoichiometric ratios ([P]0 / [C]0) that are equal to the values ​​provided in Equation 2 are also provided in Table 4.

[0159] While the numerical ranges and parameters defining the broad scope of this invention are approximate, the values ​​listed in the specific embodiments are recorded as accurately as possible. However, any value inevitably contains some error due to the standard deviation inherent in its respective measurement process, as well as the values ​​reported in the literature and experimental errors.

[0160] Therefore, the present invention is highly suitable for achieving the foregoing and inherent objects and advantages therein. Many modifications can be made by those skilled in the art that are incorporated within the spirit of the invention (partially shown in the appended claims).

Claims

1. A composition for selectively interacting with target nucleic acid molecules, characterized in that, The composition comprises: The first concentration is a first nucleic acid chain containing a first region, a second region, and a third region, wherein the second region is located between the first and third regions; The second concentration contains the second nucleic acid strand that includes the fourth and fifth regions. The target nucleic acid molecule includes the sixth and seventh regions; The first and second concentrations result in an interaction between the target nucleic acid molecule and the composition that follows Equation 1 [ΔG°] rxn =ΔG° t-TC -ΔG° nh-PC +(ΔG° v-TC -ΔG° h-PC The standard free energy determined by [P]0 is equal to the standard free energy determined by Equation 2 [-Rτln(([P]0-[C]0) / [C]0)], where the term [P]0 in Equation 2 is equal to the second concentration, the term [C]0 in Equation 2 is equal to the first concentration, and the second concentration is greater than the first concentration; and the composition has a standard free energy of hybridization with the target nucleic acid molecule of about -5 kcal / mol to about +5 kcal / mol, determined by Equation 1; and Among them, the term ΔG° in Equation 1 t-TC The standard free energy representing the hybridization between region 6 and region 1; the term ΔG° in Equation 1. nh-PC The term ΔG° represents the free energy of hybridization between the fifth and third regions; it is the term from Equation 1. v-TC The standard free energy representing the hybridization between the seventh and second regions; the term ΔG° in Equation 1. h-PC Let represent the standard free energy of hybridization between the fourth and second regions, and where ΔG° t-TC and ΔG° nh-PC The values ​​differ by more than 10%.

2. The composition according to claim 1, characterized in that, The composition also includes a label coupled to the first nucleic acid chain, wherein the label is selected from organic fluorophores, haptens, nanoparticles and radioisotopes.

3. The composition according to claim 1, characterized in that, The second concentration is approximately 1.01 times to approximately 10,000 times that of the first concentration.

4. The composition according to claim 1, characterized in that, The first or second nucleic acid chain comprises: (a) a synthetic nucleic acid analog selected from LNA, PNA, 2'-O-methyl substituted RNA, phosphate thioester substituted DNA or RNA, L-DNA, and mirror-image nucleotides; or (b) a synthetic or natural nucleotide analog, wherein the synthetic nucleotide analog is selected from inosine, 5'-nitroindole, methylated nucleotides, isocytosine and isoguanine, mirror-image nucleotides, and xDNA.

5. The composition according to claim 1, characterized in that, The second region is completely complementary to the seventh region; the first region is completely complementary to the sixth region; and / or the third region is completely complementary to the fifth region.

6. The composition according to claim 1, characterized in that, ΔG° t-TC It ranges from approximately -2 kcal / mol to approximately -16 kcal / mol.

7. The composition according to claim 1, characterized in that, The first nucleic acid strand and the second nucleic acid strand form a partially double-stranded nucleic acid probe or a partially double-stranded nucleic acid primer, wherein the first region has almost no secondary structure or no secondary structure.

8. The composition according to claim 1, characterized in that, In the minimum free energy structure calculated and evaluated under working temperature and salinity conditions, less than 50% of the nucleotides in the first region are in a double-stranded state.

9. The composition according to claim 1, characterized in that, (a) The sum of the standard free energies (G°) of the hybridization between the first region and the sixth region and between the second region and the seventh region. t-TC +ΔG° v-TC (a) More negative than -15 kcal / mol, or (b) the sum of the standard free energies of the hybridization between the third and fifth regions and between the fourth and second regions (ΔG°). nh-PC +ΔG° h-PC It is more negative than -15 kcal / mol.

10. A method for selectively amplifying multiple target nucleic acid molecules, comprising the following steps: The first and second primers are used on a sample to determine the presence or quantification of a first target nucleic acid sequence and a second target nucleic acid sequence, wherein the application of the first and second primers to the sample produces a reaction mixture, and wherein each of the first and second target nucleic acid sequences includes a sixth region and a seventh region. The first and second target nucleic acid sequences have at least five nucleotide overlaps, thereby providing an overlapping region in which the first target nucleic acid sequence differs from the second target nucleic acid sequence by one or two nucleotides. The first and second primers each comprise: The first concentration is a first nucleic acid chain containing a first region, a second region, and a third region, wherein the second region is located between the first and third regions; The second concentration includes the second nucleic acid strand containing the fourth and fifth regions; The first and second concentrations mentioned therein make the standard free energy determined by equation 2 [-Rτln(([P]0-[C]0) / [C]0))] equal to that determined by equation 1 [ΔG°] rxn =ΔG° t-TC -ΔG° nh-PC +(ΔG° v-TC -ΔG° h-PC The standard free energy determined by Formula 2, wherein the first primer interacts with the first target nucleic acid sequence and the second primer interacts with the second target nucleic acid sequence, wherein the term [P]0 in Formula 2 is equal to the second concentration and the term [C]0 in Formula 2 is equal to the first concentration; and the second concentration is greater than the first concentration; and wherein the composition has a standard free energy of hybridization with the target nucleic acid molecule of about -5 kcal / mol to about +5 kcal / mol as determined by Formula 1; and Among them, the term ΔG° in Equation 1 t-TC The standard free energy representing the hybridization between region 6 and region 1; the term ΔG° in Equation 1. nh-PC The term ΔG° represents the free energy of hybridization between the fifth and third regions; it is the term from Equation 1. v-TC The standard free energy representing the hybridization between the seventh and second regions; the term ΔG° in Equation 1. h-PC Let represent the standard free energy of hybridization between the fourth and second regions, and where ΔG° t-TC and ΔG° nh-PC The values ​​differ by more than 10%; and The reaction mixture is used in a reaction protocol sufficient to amplify the first and second target nucleic acid sequences.

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