Polymerase-Endonuclease Amplification Reaction (PEAR) and its potential applications

1. Polymerase-Endonuclease Amplification Reaction (PEAR)and its potential applications Xiaolong Wang College of Life Sciences, Ocean University of China Xiaolong@ouc.edu.cn Nucleic Acids Enzymes and Enzymes in Human Disease (NAEEHD) Jun 20, 2011, NanKai University

2. Outline • DNA amplification technologies • What is PEAR? • Potential applications of PEAR • Large-scale preparation of oligonucleotides • Preparation of repeat DNA sequences. • Detection and quantification oligonucleotides and small RNAs

3. I. DNA amplification technologies: 1. Thermocycling reactions PCR: Polymerase Chain Reaction, Science,1985 LCR: Ligase Chain Reaction, PNAS,1991 PEAR: Polymerase-Endonuclease Amplification Reaction, PLoS One, 2010 2. Isothermal reactions SDA: Strand Displacement Amplification, PNAS,1992 RCA: Rolling circle amplification, Nature Genetics,1998 LAMP: loop-mediated isothermal amplification, NAR, 2000 HDA: helicase-dependent amplification, EMBO reports, 2004 EXPAR: Exponential amplification reaction, PNAS, 2003

4. Outline • DNA amplification technologies • What is PEAR? • Potential applications of PEAR • Large-scale preparation of oligonucleotides • Preparation of repeat DNA sequences. • Detection and quantification oligonucleotides and small RNAs

5. EXPAR: Exponential amplification reaction Probe Target X Annealing X X’ R’ X’ X’ R’ X’ Annealing Strand displacement X X X’ R’ X’ Bst polymerase Nikase X R X X X Cleaving X’ R’ X’ X’ X’ • Van Ness J, Van Ness LK, Galas DJ. 2003. Isothermal reactions for the amplification of oligonucleotides, PNAS, 100 (8): 4504–4509

6. PEAR: Polymerase-Endonuclease Amplification Reaction Wang, Xu and Gou,PLoS One, 2010, 5 (1):e8430 X Annealing X X’ R’ X’ X R X R X Denaturation X’ R’ X’ Target Probe X X’ R’ X’ R’ X’ Annealing Elongation X X R X X X’ Annealing X’ R’ X’ X’ R’ X’ X’ Annealing (slipping) Cleaving Denaturation Elongation Taq polymerase X R X X’ R’ X’ Annealing Denaturation PspGI + X R X X X X R X Cleaving X’ R’ X’ X’ X’ X’ R’ X’

7. Thermostableendonuclease PspGI (NEB R0611S 1,000U 10U/uL $58.00) Dr. Shuang-yong Xu Senior ScientistNew England Biolabs Morgan R, Xiao J, Xu S (1998) Characterization of an extremely thermostable restriction enzyme, PspGI, from a Pyrococcus strain and cloning of the PspGI restriction-modification system in Escherichia coli. Appl Environ Microbiol 64(10): 3669–3673.

8. +++++---- HLH--HH-- ++-+-+-+- Target: PspGI: Polymerase: M123456789 bp 100 40 20 10 Input target concentration (nM) 110-1 10-210-310-4 10-510-6 M1234567 bp 100 20 10

9. Outline • DNA amplification technologies • What is PEAR? • Potential applications of PEAR • Large-scale preparation of oligonucleotides • Preparation of repeat DNA sequences. • Detection and quantification oligonucleotides and small RNAs

10. Many diseases are caused by genes

11. Gene therapy a possible solution

12. Repairing a damaged good gene

13. Inhibiting a bad or overexpressed gene • Gene knockout • Gene Knockdown • mRNA • RNAi • miRNA • Antisense oligonucleotides • miRNA • Antisense oligonucleotides • miRNA sponge

14. RNAiis effective for mRNA knockdown

15. miRNA is a endogenous small RNA

16. How miRNA works?

18. Antisense miRNA oligonucleotide (AMO) J Weiler et al Anti-miRNA oligonucleotides Gene Therapy (2006) 13, 496–502

19. * Antisense oligonucleotide (ASO)

20. Fomivirsen (Vitravene) — the first and only antisense antiviral drug approved by FDA $63.87 USD Fomivirsen (ISIS 2922)

21. Modified ASO Chemically modified oligonucleotide analogs that have been used as anti-miRNA inhibitors (AMOs).

22. Locked Nucleic Acid (LNA)

23. JesperWengel Professor, University of Southern Denmark JesperWengel is professor of bioorganic chemistry at the University of Southern Denmark and Director of the Nucleic Acid Center, a research center of excellence focused on nucleic acid chemical biology. He is the co-inventor of LNA (locked nucleic acid) and inventor of UsiRNA, and he is co-founder of RiboTask, a biotech company focused on developing and marketing novel technologies for gene silencing.

24. Locked Nucleic Acid (LNA) Wahlestedt C et al. PNAS 2000;97:5633-5638

25. SCIENCE VOL 327 8 JANUARY 2010

28. Whyamplify oligonucleotides? • Almost all oligonucleotides are chemical synthesized, but large-scale synthesis of oligonucleotides is still difficult, • 1. Purity: n-1 deletions and failure sequences; • 2. Large-scale HPLC purification is difficult; • 3. Uses hazardous Chemicals; • 4. Expensive instruments.

29. Chemical Synthetic modified AntisenseOligonucleotides is extremely expensive!

32. A possible way out: PEAR for large-scale Enzymatic Production of Antisense Oligonucleotides

36. PEAR for Enzymatic Production of Oligos

38. Is it feasible to synthesize modified ASO by PEAR?

39. Outline • DNA amplification technologies • What is PEAR? • Potential applications of PEAR • Large-scale preparation of oligonucleotides • Preparation of repeat DNA sequences. • Detection and quantification oligonucleotides and small RNAs

40. Potential applications of Repeat DNA • PEAR is a minimal DNA replication system, to study the origin and evolution of repetitive DNA in genome, as well as the origin and evolution of genetic material and life. • The repeat PEAR product DNA can be transferred into cells or organisms to study • Function of repeat DNA sequences. • Synthetic biology • Molecular evolution

41. Target X Annealing Probe X X’ R’ X’ Denaturation X’ R’ X’ X R X R X Denaturation Annealing X X’ R’ X’ R’ X’ Elongation X X R X X X’ Annealing X’ R’ X’ X’ R’ X’ X’ Annealing (slipping) Cleaving X R X Elongation dNTPs Taq polymerase Denaturation X’ R’ X’ Annealing Denaturation X R X X X PspGI X R X + Cleaving X’ R’ X’ X’ X’ X’ R’ X’

42. Outline • DNA amplification technologies • What is PEAR? • Potential applications of PEAR • Large-scale preparation of oligonucleotides • Preparation of repeat DNA sequences. • Detection and quantification oligonucleotides and small RNAs

43. I. Real-time detection methods 1. Thermocycling reactions PCR: Polymerase Chain Reaction LCR: Ligase Chain Reaction PEAR: Polymerase-Endonuclease Amplification Reaction 2. Isothermal reactions SDA: Strand Displacement Amplification RCA: Rolling circle amplification LAMP: loop-mediated isothermal amplification HDA: helicase-dependent amplification EXPAR: Exponential amplification reaction

44. m Target miRNA Poly A Tailing PAP m (A)nAAAAA Reverse transcription Oligo dT dNTPs Reverse transcriptase m (A)nAAAAA TTTTTTTT cDNA M’ Universal Tag RNase H TTTTTTTT cDNA M’ Universal Primer Gene Specific Primer Real-time PCR

45. Target miRNA m Poly A Tailing PAP m (A)nAAAAA Reverse transcription Oligo dT dNTPs Reverse transcriptase m (A)nAAAAA TTTTTTTT cDNA M’ RNase H TTTTTTTT cDNA M’ Repeat Probe Real-time PEAR

46. Probe Target miRNA m M’ R’ M’ Denaturing Annealing m M’ R’ M’ E. Coli Polymerase I M R m M’ R’ M’ Repeat Probe Real-time PEAR

47. Labeling PEAR probe and real-time PEAR PspGI X X R X X X X’ R’ X’ PspGI X’ R’ X’ Taq polymerase X’ X’ TAMRA FAM FAM TAMRA FAM TAMRA Fluorescent intensity Input target concentration (nM) 110-1 10-210-310-4 10-510-6 M1234567 bp 100 20 10 Number of Cycles

48. Why thermocycling reaction (PCR) outperforms isothermal reactions? PCR is much more reliable in most situations: PCR Reaction is tightly controlled by thermocycling, easy and reliable, while isothermal reaction is not controllable once started; Thermocycling helps mixing reaction mixture; Instrument is not a real problem, especially in a modern molecular biology laboratory;

49. Conclusion • PEAR is a new DNA amplification technology • PEARis Potentially useful for: • Large-scale preparation of oligonucleotides • Preparation of repeat DNA sequences. • Detection and quantification oligonucleotides and small RNAs

50. Acknowledgements Dr. Shuang-yong Xu, Senior ScientistRestriction EnzymesNew England Biolabs 苟德明 博士、教授 深圳大学特聘教授 Assistant Professor, University of Illinois at Chicago