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KR-20260068002-A - A method for simulating a toehold-mediated rolling circle amplification and an optimized composition for rolling circle amplification reaction

KR20260068002AKR 20260068002 AKR20260068002 AKR 20260068002AKR-20260068002-A

Abstract

The present invention relates to a tohold-mediated rotary ring amplification simulation method and an optimized rotary ring amplification reaction composition, and more specifically, to a tohold-mediated rotary ring amplification simulation method comprising: (a) a step of constructing a TRCA simulation model by applying an amplification reaction rate function, an ion concentration function, an addition reaction function, and a concentration change function; and (b) a step of performing TRCA simulation by substituting miRNA, a dumbbell DNA template (DBTP), a reverse primer (Pr), a cutting enzyme (nE), dNTPs, DNA polymerase (DNAP), Tris, and Mg 2+ concentrations; The present invention relates to a composition for a rotary ring amplification reaction comprising a dumbbell DNA template (DBTP), dNTP, DNA polymerase (DNAP), and Mg²⁺ , wherein the concentration ratio of the DBTP, dNTP, DNAP, and Mg²⁺ is 10³ -10⁴ : 10⁶ -2× 10⁷ :1-15 : 10⁷ -10⁸ .

Inventors

  • 김은정
  • 구보람
  • 이지희

Assignees

  • 인천대학교 산학협력단
  • 전남대학교산학협력단

Dates

Publication Date
20260513
Application Date
20251103
Priority Date
20241104

Claims (12)

  1. A toehold-mediated rolling circle amplification (TRCA) simulation method including the following steps: (a) a step of constructing a TRCA simulation model by applying an amplification reaction rate function, an ion concentration function, an addition reaction function, and a concentration change function; and (b) Step of simulating TRCA by substituting miRNA, dumbbell DNA template (DBTP), reverse primer (Pr), cutting enzyme (nE), dNTP, DNA polymerase (DNAP), Tris and Mg 2+ concentrations.
  2. Method according to claim 1, characterized in that the amplification reaction rate function is defined by the following mathematical formulas 11 to 13 based on the following reaction formulas 1 to 3: [Reaction Equation 1] [Reaction Equation 2] [Reaction Equation 3] [Mathematical Formula 11] [Mathematical Formula 12] [Mathematical Formula 13] In the above reaction formulas 1 to 3 and mathematical formulas 11 to 13, The above mDB is a complex of miRNA and DBTP, and The above E is DNA polymerase (DNAP), and The above mDB·E is a composite of mDB and E, and The above S is a dNTP, and The above DP is a complex of DNA and Pr, and The above DP·E is a complex of DP and E, and The above DNA br is branched DNA, and The above N is a cutting enzyme, and The above D·N is a complex of DNA and N, and The above DNA nk is a truncated DNA, and The above V DNA , V DNAbr and V DNAnk are DNA, DNA br, and DNA nk, respectively. It is the synthesis rate, and [mDB], [S], [Mg 2+ ], [DP], and [D] are the concentrations of mDB, S, Mg 2+ , DP, and DNA, respectively, and [E] 0 , [Pr] 0, and [N] 0 are the initial concentrations of E, Pr, and N, respectively, and k 1 , k 2 , k -2 , k 3 , k 4 , k 5 , k -5 , k 6 , k 7 , k -7 and k8 are reaction rate constants for the reactions corresponding to Equations 1 to 3 , respectively, and K Mg is the Michaelis-Menten constant for Mg 2+ in DNAP.
  3. Method according to claim 2, characterized in that the ion concentration function is defined by the following mathematical formulas 14 to 28: [Mathematical Formula 14] [Mathematical Formula 15] [Mathematical Formula 16] [Mathematical Formula 17] [Mathematical Formula 18] [Mathematical Formula 19] [Mathematical Formula 20] [Mathematical Formula 21] [Mathematical Formula 22] [Mathematical Formula 23] [Mathematical Formula 24] [Mathematical Formula 25] [Mathematical Formula 26] [Mathematical Formula 27] [Mathematical Formula 28] In the above mathematical formulas 14 to 28, [MgNTP 2- ], [Mg 2+ ], [NTP 4- ], [Mg 2 NTP], [Mg 2+ ], [MgNTP 2- ], [MgHNTP - ], [HNTP 3- ], [MgPPi 2- ], [PPi 4- ], [Mg 2 PPi], [MgHPPi - ], [HPPi 3- ], [H + ], [H 2 PPi 2- ], [HTris + ] and [Tris] are MgNTP 2- , Mg 2+ , NTP 4- , Mg 2 NTP, Mg 2+ , MgNTP 2- , MgHNTP - , HNTP 3- , MgPPi 2- , PPi 4- , Mg 2 PPi, respectively. The concentrations of MgHPPi- , HPPi 3- , H + , H2PPi 2- , HTris +, and Tris are, [Mg] 0 , [dNTP] 0 , [PPi 4- ] 0 , [Tris] 0 , [H + ] 0 are the initial concentrations of Mg, dNTP, PPi 4- , Tris, and H +, respectively, and K MgNTP2- , K Mg2NTP , K MgHNTP- , K HNTP3- , K MgPPi2- , K Mg2PPi , K MgHPPi- , K HPPi3- , K H2PPi2- , and K Tris+ are equilibrium constants corresponding to Equations 14 to 23, respectively.
  4. Method according to claim 3, characterized in that the addition reaction function is defined by the following mathematical formulas 29 and 30 to 34 based on the following reaction formula 4: [Reaction Equation 4] [Mathematical Formula 29] [Mathematical Formula 30] [Mathematical Formula 31] [Mathematical Formula 32] [Mathematical Formula 33] [Mathematical Formula 34] In the above reaction formula 4 and the above mathematical formulas 29 to 34, The above k precip is the precipitation reaction rate constant, and The above V precip is the precipitation reaction rate, and [Mg 2 PPi] is the concentration of Mg 2 PPi, and [Mg 2 PPi] eq is the concentration of Mg 2 PPi at equilibrium, and The above V deact,E is the DNAP inactivation rate, and The above k deact,E is the DNAP inactivation rate constant, and The above [E] 0 is the initial concentration of DNAP, and The above V deact,N is the cleavage enzyme inactivation rate, and The above k deact,N is the cleavage enzyme inactivation rate constant, and The above [N] 0 is the initial concentration of the cutting enzyme, and V deg,DNA, V deg,DNAbr and V deg and DNAnk are the degradation reaction rates of DNA, DNA br, and DNA nk , respectively, and k Ac , k Ba , and k Mg are the rate constants for the degradation of DNA by acid, base, and magnesium; k Ac,b , k Ba,b, and k Mg,b are the rate constants for the degradation of DNA br by acid, base, and magnesium; and k Ac,n , k Ba,n, and k Mg,n are the rate constants for the degradation of DNA nk by acid, base, and magnesium. [H + ], [ OH- ], [Mg2 + ], [DNA], [ DNA br ], and [ DNA nk ] are the concentrations of H + , OH- , Mg2 + , DNA, DNA br, and DNA nk , and n ac , n ba, and n mg are the orders of DNA degradation reactions by acid, base, and magnesium, and n DNA , n DNAbr and n DNAnk are the degradation reaction orders for the concentration dependence of DNA, DNA br , and DNA nk , respectively.
  5. A method according to claim 4, characterized in that the concentration change function is defined by the following mathematical formulas 35 to 50: [Mathematical Formula 35] [Mathematical Formula 36] [Mathematical Formula 37] [Mathematical Formula 38] [Mathematical Formula 39] [Mathematical Formula 40] [Mathematical Formula 41] [Mathematical Formula 42] [Mathematical Formula 43] [Mathematical Formula 44] [Mathematical Formula 45] [Mathematical Formula 46] [Mathematical Formula 47] [Mathematical Formula 48] [Mathematical Formula 49] [Mathematical Formula 50] In the above mathematical formulas 35 to 50, [Mg 2+ ] 0 , V precip , [dNTP] 0 , V DNA , V DNAbr , V DNAnk , [PPi 4- ] 0 , [Tris] 0 , [H+] 0 , k 1 , [miRNA], [E] 0 , V deact,E , [mDB], [DNA], V deg,DNA , [DP], k 4 , [N] 0 , V deact,N , V deg,DNAbr and V deg,DNAnk are as defined in any one of claims 1 to 4, and [DBTP], [P], [DBTP·E], [E], [DNA br ], and [DNA nk ] are the concentrations of DBTP, P, DBTP·E, E, DNA br, and DNA nk , respectively, where P, DBTP·E, E, DNA br , and DNA nk are as defined in paragraph 1, and k a and k d are the forward and reverse reaction rate constants of DBTP and E, respectively, and v dNTP is the number of dNTPs required.
  6. In paragraph 5, the method is characterized by additionally including the following steps after step (a) and before step (b): (a') A step of calculating optimal parameter values by comparing the simulation results derived by substituting arbitrary parameters into the TRCA simulation model constructed in step (a) with the actual TRCA DNA amplification results; and (a'') A step of applying parameter values calculated in step (a') above to a TRCA simulation model; Here, the above parameters are K Mg , k deact,E , k deact,N k 1 , k 2 , k -2 , k 3 , k 4 , k 5 , k -5 , k 6 , k 7 , k -7 , k 8, k Ac , k Ba , k Mg , k Ac,b , k Ba,b , k Mg,b , k Ac,n , k Ba,n and k Mg,n are.
  7. Method according to claim 6, wherein the above step (a') is performed through a function defined by the following mathematical formula 51: [Mathematical Formula 51] In the above mathematical formula 51, i is an index representing the number of experiments, N is the total number of experiments, and j is the index of the time-based data point for each experiment, T i is the total number of data points over the i-th experiment, and k is an index representing the type of concentration variable, and C i is the total number of concentration variables in the i-th experiment, and x is a vector consisting of all parameters, and Y exp is the actual total DNA concentration, and Y sim is the total DNA concentration from the simulation results.
  8. A composition for a rotary ring amplification reaction comprising a dumbbell DNA template (DBTP), dNTP, DNA polymerase (DNAP), and Mg²⁺ , wherein the concentration ratio of the DBTP, dNTP, DNAP, and Mg²⁺ is 10³ -10⁴ : 10⁶ -2× 10⁷ :1-15 : 10⁷ -10⁸ .
  9. In claim 8, the dumbbell DNA template is a dumbbell-structured nucleic acid template comprising one stem sequence and two loop sequences, A composition for a rotary ring amplification reaction characterized by having a toehold site, a cleavage enzyme recognition site, and a reverse primer binding site located on the above loop sequence.
  10. A composition for a rotary ring amplification reaction according to claim 8, wherein the composition further comprises a cutting enzyme, and the concentration ratio of the DBTP, dNTP, DNAP, Mg 2+ , and cutting enzyme is 10³ -10⁴ : 10⁶ -2× 10⁷ :1-15 : 10⁷ -10⁸ :10⁻500.
  11. A composition for a rotary ring amplification reaction according to claim 8, wherein the composition further comprises a reverse primer, and the concentration ratio of the DBTP, dNTP, DNAP, Mg 2+ and the reverse primer is 10³ -10⁴ : 10⁶ -2× 10⁷ : 1-15 : 10⁷ -10⁸ : 10³ -10⁴ .
  12. A composition for a rotary ring amplification reaction according to claim 8, wherein the composition further comprises a cutting enzyme and a reverse primer, and the concentration ratio of the DBTP, dNTP, DNAP, Mg²⁺ , cutting enzyme and reverse primer is 10³ -10⁴ : 10⁶ -2× 10⁷ :1-15 : 10⁷ -10⁸ :10-500 : 10³ -10⁴ .

Description

A method for simulating a toehold-mediated rolling circle amplification and an optimized composition for a rolling circle amplification reaction The present invention relates to a tohold-mediated rotary ring amplification simulation method and an optimized rotary ring amplification reaction composition, and more specifically, to a tohold-mediated rotary ring amplification simulation method comprising: (a) a step of constructing a TRCA simulation model by applying an amplification reaction rate function, an ion concentration function, an addition reaction function, and a concentration change function; and (b) a step of performing TRCA simulation by substituting miRNA, a dumbbell DNA template (DBTP), a reverse primer (Pr), a cutting enzyme (nE), dNTPs, DNA polymerase (DNAP), Tris, and Mg 2+ concentrations; The present invention relates to a composition for a rotary ring amplification reaction comprising a dumbbell DNA template (DBTP), dNTP, DNA polymerase (DNAP), and Mg²⁺ , wherein the concentration ratio of the DBTP, dNTP, DNAP, and Mg²⁺ is 10³ -10⁴ : 10⁶ -2× 10⁷ :1-15 : 10⁷ -10⁸ . Gene amplification systems enable the detection and analysis of nucleic acids and have established themselves as fundamental tools in the fields of molecular biology and diagnostics. Quantitative polymerase chain reaction (qPCR), first developed in the 1990s, has rapidly evolved and is now the standard for laboratory-based nucleic acid analysis and detection (Non-Patent Literature 1). However, due to various limitations, the application of qPCR in the field of point-of-care (POC) diagnostics has been restricted. The biggest limitation is the dependence on expensive thermal cyclers, which require expensive equipment and a stable power supply because optical signals must be read under precise temperature control. Due to these characteristics, it lacks portability and is unsuitable for various POC applications. To address this, isothermal amplification techniques capable of operating at a single temperature have been developed, and efforts have been made to simplify the thermal cycling process. A representative example is rolling circle amplification (RCA), a method in which DNA polymerase (DNAP), possessing strong strand displacement activity, amplifies DNA by rotating along a circular DNA template at a constant temperature. RCA can be performed not only in solution but also on solid substrates such as glass, plastic, metal, and nano/microparticle surfaces, and is applicable in complex biological environments such as cell membranes and intracellular compartments. Thanks to this robustness, versatility, and programmability, RCA is widely used in in vitro diagnostics, biosensors, and bioanalysis, enabling sensitive detection of DNA, RNA, proteins, small molecules, and cells. Meanwhile, although RCA technology is widely used in the fields of bioanalysis and biosensors, systematically determining optimal conditions remains a challenge. A typical RCA reaction requires key components such as a cyclic DNA template, primers, DNA polymerase, deoxynucleoside triphosphate (dNTP), and a reaction buffer containing Mg²⁺ . The concentrations of these key components significantly affect the overall amplification efficiency. To optimize these RCA and their variant technologies for POC diagnosis or quantitative analysis, an in-depth understanding of fundamental kinetic parameters, including the initial rate and final yield, is essential. In particular, optimizing the initial amplification rate is crucial for shortening detection time and reducing costs, while ensuring reliability by increasing the final yield is key for detecting targets at low concentrations. Variants of RCA, such as HRCA and NRCA in the present invention, exhibit complex reaction kinetics because amplification is initiated simultaneously at multiple locations. Accordingly, it is important to quantitatively understand the influence of the concentration and binding efficiency of each component on the initial rate and final yield. Figure 1 illustrates the reaction pathways of conventional TRCA and modified TRCA (HTRCA, NTRCA, HNTRCA). Figure 2 shows the results of confirming target miRNA-specific DBTP circularization, Figure 2a shows the enzymatic self-linking of linear template DNA, and Figure 2b shows the results of 15% native and denatured PAGE analysis to confirm DBTP circularization in the presence of target miRNA. Figure 3 shows the results of the analysis of the DBTP melting curve in a TRCA reaction buffer solution without DNAP, where Figure 3a shows the melting curve of DBTP and Figure 3b shows the derivative curve corresponding to Figure 3a. Figure 4 shows the real-time fluorescence curves, initial reaction rates, and fluorescence signals after the reaction is finished under various conditions of TRCA, where Figure 4a shows the results of performing TRCA with different concentrations of DBTP, Figure 4b shows the results of performing TRCA with different concentrations of Mg,