13 1 Vol13 No1 29 2 Life Science Research Feb 29 29 *,,,,, 4182 :,, (Molecular Beacon, MB),,,, 5~5 U / ml, 5 U / ml, Alu : ; ; : Q55 : A : 17-7847(29)1-6-5 A Novel Fluorescence Assay for the Activity of Restriction Endonuclease Based on Molecular Beacon HUO Xi-qin YAN Hong-fei LI Jun ZHOU Xing-wang LI Wei YANG Xiao-hai * State Key Laboratory of Chemo / Biosensing and Chemometrics College of Chemistry and Chemical Engineering Biomedical Engineering Center Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province Hunan University Changsha 4182 Hunan China Abstract A novel and simple fluorescence assay for the activity of Xsp restriction endonuclease was proposed based on molecular beacons cutting by restriction endonuclease The temporary and dynamic double DNA structure was suggested as the possible cleavage mechanism Under the optimal condition the initial velocity was proportional to the concentration of endonuclease in the range of 5 to 5 U / ml and the detection limit was 5 U / ml This method can also be extended to other restriction endonucleases such as Alu by changing the reorganization sequence in the loop part of MB Molecular Beacon MB Key words molecular beacon fluorescence assay restriction endonucleases Life Science Research 29 13 1 6~1 [1] [2] λdna : 28-1-25 : 28-12-5 : 9663 28FJ325 3SSY16 8JJ12 : 1957- * 1971- E-mail yangxiaohai@hnucn Tel 731-882393 Fax 731-882393
1 : 7 1 11 LS-55 Perkin Elmer Molecular Beacon Thermo Neslab MB MB1 MB2 [3~6] Li [7] DNA 1 5 - TAMRA 3 - DABCYL MB1 Biggins [8,9] CTAG Xsp Alu AGCT 4 N1 MB1 N2 MB1 4 N3 N3 MB1 N4 N4 MB1 1 Alu Takara TEMED Sigma St LouisMO Alu MB2 Name MB1 MB2 N1 N2 N3 N4 1 Table 1 The sequence of molecular beacon and other oligonucleotides 5 - TAMRA -CGTTGA CAG ACA TGC CTAG ACA TGC CT TCAACG- DABCYL -3 5 - TAMRA -CGTTGA CAG ACA TGC AGCT ACA TGC CT TCAACG- DABCYL -3 5 -TGT CTA GGC A-3 5 -CAT GTA GCT GCA TG-3 5 -AGG CAT GTC T-3 5 -AGG CAT GTC TG-3 Sequence 12 Perkin Elmer LS- 55 37 558 nm 58 nm 5 1 nm 13 1 2 mmol / L Tris-HCl ph 85 1 mmol / L MgCl 2 1 mmol / L KCl 1 mmol / L DTT MB1 MB2 2 μl 12 5 μmol / L MB1 DNA 5 nmol / L 1 U Xsp
8 29 2 μl 9 min 65 5 μl 2 % MB1 1 TBE ph = 8 22 V 3 h 1 5 mg / L 3 min 2 21 25 15 DNA 5 DNA 5 1 1 5 2 DNA [1~12] 2 Fig2 Real-time monitoring cleavage process DNA 1 3A 1~3 MB1 MB1 N1 MB1 N2 [7] N1 MB1 1 A N1 N2 N3 N4 B 1 (A),, ; (B), DNA (N1-N4) Fig1 Principle of the cleavage process based on molecular beacon A The instantaneous dimmer MB formed at the cleavage site then the MB was cut by endonuclease and fluorescence restored B The study of cleavage mechanism by adding different oligonucleotides N1-N4 to form different hybrids with MB N1 MB1 MB1 N2 Xsp 3B N3 N4 MB1 N4 N3 1 22 4 MB1 4 2 1 1 Xsp 2 MB1 MB1 N1 MB1 N1 2 3 1 MB1 MB N1 min MB1 6 Fluorescence intensity 4 35 3 2 1 t
1 : 9 ΔF 1 8 6 A 2 1 3 4 4 2 2 2 3 5 1 1 5 2 5 1 1 5 2 3 (A) (1) MB1,(2) (3) MB1 N1 N2 ; (B) (1) MB1,(2) (3) MB1 N3 N4 Fig3 The effect of recognition site structure on the cleavage reaction A Time scan curves 1~3 represent the MB1 and hybrids of MB1 with oligos N1 or N2 B Time scan curves 1~3 represent the MB1 and hybrids of MB1 with double oligos N3 or N4 ΔF 1 8 6 B 1 3 MB1 MB1 N1 Mg 2 1 mmol / L MB1 MB1 4 25 3 2 MB1 15 DNA 1 5 1 2 3 4 5 Mg 2 MB1 4 Fig5 The effect of Mg 2 concentration on the cleavage 1: MB1; 2: MB1 N1; 3: MB1 N1 ; 4: MB1 velocity of MB1 6 min Fig 4 Image of gel electrophoresis 24 1 MB1 2 MB1 and oligos N1 3 MB1 oligos N1 and Xsp endonuclease 4 MB1 and Xsp endonuclease Samples were incubated for 6 min first The direction is from the top to the end Relative vetocity 1 2 3 4 5 6 Mg 2 / mmol L -1 23 Mg 2 MB1 ~ Mg 2 Xsp 5 nmol / L 5 5 Mg 2 MB1 6 MB1 5 Mg 2 1~1 mmol / L nmol / L Mg 2 Mg 2 1 mmol / L MB1 5 nmol / L Mg 2 MB1 MB1 5 mmol / L Xsp MB1 Mg 2 Mg 2 1 / V-1 / [S] V [S]
1 29 MB1 6 6 1 / V 1 / [S] C-T-A-G MB1 A-G-C-T Alu MB1 3 MB2 25 Alu 2 Alu 25 MB1 MB1 5 nmol/l 7 23 747A-753A Xsp 5~5 U / ml 216-223 V =3811 C V s -1 C U / ml 5 U / ml Fluorescence 3 25 2 15 1 5 V/s -1 Cleavage velocity / s -1 15 1 5 1 /V/ Flu -1 s 35 3 25 2 15 1 5-5 1 2 3 4 5 6 1 /[S] nmol L -1 5 1 1 5 2 MB concentration nmol L -1 25 2 15 1 5-5 1 2 3 4 5 6 Endonuclease concentration / U ml -1 8~4 U / ml (References): [1] JELTSCH A WENZ C WENDE W et al Engineering novel 6 restriction endonucleases principles and applications [J] Trends in Biotechnology 1996 14 7 235-238 Fig6 The effect of MB concentration on the cleavage [2] SAMBROOK J Molecular Cloning A Laboratory Manual[M] velocity Inset is the Lineweaver-Burke plot for 1 / V~1 / [S] New York Cold Spring Harbor Laboratory Press 1989 73-75 [3] TYAGI S KRAMER F R Molecular beacons probes that fluoresce upon hybridization[j] Nature Biotechnology 1996 14 3 33-38 [4] FANG X LI J J PERLETTE J et al Molecular beacons novel fluorescent probes[j] Analytical Chemistry 2 72 [5] YAO G TAN W Molecular-beacon-based array for sensitive DNA analysis[j] Analytical Biochemistry 24 331 2 [6] TAN W WANG K DRAKE T J Molecular beacons [J] Current Opinion in Chemical Biology 24 8 5 547-553 [7] LI J J GEYER R TAN W Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of singlestranded DNA[J] Nucleic Acids Research 2 28 11 e52 [8] BIGGINS J B PRUDENT J R MARSHALL D J et al A continuous assay for DNA cleavage The application of break lights to enediynes iron-dependent agents and nucleases[j] Proceedings of the National Academy of Sciences 2 97 25 13537-13542 [9] [J] ZHANG Yong-you LI Qing-ge LIANG Ji-xuan et al Hairpin probes for real-time assay of restriction endonucleases[j] Acta Biochimica et Biophysica Sinica 22 34 3 329-332 [1] NISHIGAKI K KANEKO Y WAKUDA H et al Type II 2 4 6 8 1 restriction endonucleases cleave single-stranded DNAs in gene ral[j] Nucleic Acids Research 1985 13 16 5747-576 7 Xsp I - [11] HOFER B RUHE G KOCH A et al Primary and secondary, Xsp I 5 5 25 5 25 5 U/mL; structure specificity of the cleavage of single-stranded DNA : Xsp I by endo-nuclease HinfI[J] Nucleic Acids Research 1982 1 Fig7 Fluorescence time scan curves of MB cleaved by different concentration of 9 2763-2773 The concentrations of are 5 5 25 5 25 and [12] MUCKE M KRUGER D H REUTER M Diversity of Type II 5 U/mL from bottom to top Inset is the linear relationship restriction endonucleases that require two DNA recognition of the initial cleavage velocity and the concentration of sites[j] Nucleic Acids Research 23 31 21 679-684