Development Of A Novel Nucleic Acid Detection Method Based On ATP-Releasing Nucleotide Probe

ATP-releasing nucleotides(ARNs)can replace natural deoxynucleotides to perform primer extension reactions catalyzed by DNA polymerase,and ATP is generated during the primer extension process.Combining ARNs with the existing nucleic acid detection signal amplification technology can achieve ultra-sensitive detection of targets by detecting ATP molecules.The combination of enzyme-assisted detection signal amplification strategy and molecular beacon can realize the cascade amplification of detection signals.Based on the above content,this paper uses the principle of "bridged" ATP to convert it into a fluorescent signal or a bioluminescence signal,and designs two biosensing systems to achieve ultra-sensitive detection of target substances.(1)The paper developed a new method of synthesizing ARNs,which avoids the shortcomings of introducing ATP through raw materials,and synthesized three kinds of nucleotide molecular probes by this method,namely dGP4A and dAP4A,dTP4A.(2)Taking advantage of the property that ATP is the coenzyme of T4 DNA ligase,combining ARNs with molecular beacons,T4 DNA ligase,and polymerase to construct an ultra-sensitive method for detecting ATP.In the existing method,T4 DNA ligase uses ATP to connect two short DNA fragments to open the hairpin structure of the molecular beacon and generate a fluorescent signal.In this way,in theory,one molecule of ATP can generate one molecule of fluorescent signal.We add ARNs raw materials to the above reaction system,perform enzymatic reaction under the action of DNA polymerase,and release ATP at the same time,and the generated ATP will continue to be used in the reaction of T4 DNA ligase to open more molecular beacons.Through the above design,an ATP circulation system is realized,and the exponential amplification of the ATP quantity is realized by polymerase.In theory,the system can open all molecular beacons with one ATP molecule.The experimental results show that the detection sensitivity of this method has reached 2.29 pM,which is nearly two orders of magnitude higher than that of the existing methods,and it has realized the ultra-sensitive detection of ATP.(3)Combining ARNs with strand displacement amplification technology and hairpin structure,under the catalysis of DNA polymerase,a biosensing system that can be used for ultra-sensitive detection of nucleic acid strands is constructed.The sensor system is designed with two probes of hairpin structure HP1 and HP2,and the 3'ends of HP1 and HP2 can be paired with complementary bases.When the target chain exists,HP1(or HP2)specifically binds to it and is in an open state.The opened HP1(or HP2)hybridizes with HP2(or HP1)to form a ternary complex.Continuous strand displacement reaction catalyzed by DNA polymerase;using ARNs as the raw material for the above reaction,ATP is produced in the system after the reaction,and then used ATP bioluminescence method(luciferase and luciferin)reads the detection signal.The combination of the above-mentioned sensing system and nucleic acid aptamer technology further realizes the sensitive detection of aflatoxin B1(AFB1).