Artificial Nucleic Acids -Their Developments and Recent Applications

Transcription

1 Artificial Nucleic Acids -Their Developments and Recent Applications Bioorganic Chemistry Laboratory D2 Kenichiro Ito Organic Seminar 2012/5/7 1

2 Nucleic acids play central roles in life Replication Transcription Translation DNA (Reverse Transcription) mrna (RNA) Protein Central Dogma DNA Forms stable duplex by the Watson- Crick base pairing RNA Basically single-stranded (Also can form higher structure) 2

3 Targeting DNA DNA stores genetic information in vivo Mutation in genomic DNA can cause genetic diseases Genomic DNA Probe Detecting mutations in genomic DNA prediction of genetic diseases Wild type genome Mutated genome Easy detection of mutations in DNA 3

4 Targeting mrna Normal mrna Translation Defective mrna Antisense oligonucleotides -hybridize and block translation Normal proteins Defective proteins Diseases mrna is a potent drug target for therapeutics sirna oligonucleotides RISC Degradation -induce RNAi with RISC protein complex and degrade targeted RNA 4

5 Structures and interactions of DNA and RNA DNA Adenine (A) Thymine (T) RNA Adenine (A) Uracil (U) Phosphodiesterbackbone Cytosine (C) Guanine (G) Cytosine (C) Guanine (G) Recognitions of DNA and RNA are defined by base pairings of Watson-Crick s rule Easy to design nucleic acids-based targeting molecules 5

6 Tools for DNA/RNA targeting Nuclease resistance Sequence recognition Recognition of mismatched base pair Natural nucleic acids Poor (Natural phosphodiester linkage) Fixed (Four general bases) Normal Artificial nucleic acids Good (non-natural backbone) Tunable (modified base) Good (flexibility of backbone) Toxicity Low Low (*up to the modification) Artificial nucleic acids are more useful tools than natural ones in DNA/RNA targeting in vivo and in vitro 6

7 Table of contents Base modifications ( Tuning base pairing) Backbone modifications 1. PNA (Peptide nucleic acid) 2. BNA (Bridged nucleic acid) 3. UNA (Unlocked nucleic acid) 7

8 Base modification- G-Clamp G-Clamp: Cytosine nucleobase with 9-(2-guanidinoethoxy) phenoazide G C G:C 3 hydrogen bonds K. Lin and M. D. Matteucci, J. Am. Chem. Soc. 1998, 120, C. J. Wilds et al. Angew. Chem. Int. Ed. 2002, 41, 115. G-Clamp G:G-Clamp 5 hydrogen bonds 1 substitution of C to G-Clamp Tm (10 bp) = +18 C G 8

9 Base modification Us and D for PNA T A A:T 2 hydrogen bonds 2-thiouracil (Us) J. Lohse et al. Proc. Natl. Acad. Sci. USA, 1999, 96, ,6-diaminopurine (D) G. Haaima et al. Nucleic Acids Res. 1997, 25, Incorporated into PNA Stabilization of PNA/DNA duplex Destabilization of PNA/PNA duplex A D Us T Us A:Us 2 hydrogen bonds D:T 3 hydrogen bonds Higher affinity to natural nucleic acids, Avoiding self-duplex forming of PNA D D:Us 2 hydrogen bonds Steric hindrance 9

10 Backbone modification #1: PNA- peptide nucleic acid PNA: DNA mimic with poly[n-(2-aminoethyl)glycine] backbone DNA PNA Phosphodiester bond Amide bond PNA is charge-neutral, while DNA has negative charges P. E. Nielsen, et al. Science. 1991, 254, DNA/PNA has no electrostatic repulsion and more stable than DNA/DNA duplex PNA is resistant to degradation by nucleases 10

11 DNA detection using PNA probes- Molecular beacon PNA Molecular beacon (for detection of a point mutation in DNA) DABCYL (quencher) Fluorescein 1. Wild-type DNA 2. DNA with a mutation DNA recognition sequence Hybridization Gap-cleaving enzyme S. Ye et al. Anal. Biochem. 2007, 363, 300. Fluorescence Quench

12 Backbone modifications of PNA Further modifications of PNA backbone have been achieved. PNA (Standard) Positive charges and chirality Lys-PNA (D-Lysine side chain) Rigidity Cyp-PNA (trans-cyclopentyl linkage) Backbone is fixed in the best angle by the linkage lower entropy cost Electrostatic stabilization of PNA/DNA duplex D-lysine can give rigidity of right-handed helical structures for DNA/PNA duplex S. Sforza et al. Eur. J. Org. Chem. 2000, J. K. Pokorski et al, J. Am. Chem. Soc. 2004, 126,

13 Artificial nucleic acids for RNA targeting in vivo For in vivo use, artificial nucleic acids need Low-toxicity High resistance to nucleases High binding affinity to DNAs or RNAs Easy transfection Being recognized by several proteins (PNA) Improvement is necessary for in vivo usage RNA is more potent target in vivo 1.Antisense oligonucleotide 2.siRNA oligonucleotide a 1.BNA (Bridged nucleic acid) 2.UNA (Unlocked nucleic acid) 13

14 Backbone modification #2: BNA- bridged nucleic acid 2,4 -BNA: 2 -O, 4 -C-methylene- -D-ribofuranosyl monomer 2,4 -BNA DNA C2 -endo C3 -endo RNA Locked in C3 -endo pucker J. Wengel, Acc. Chem. Res. 1998, 32, 301. S. Obika Tetrahedron Lett. 1998, 38, C2 -endo C3 -endo 14

15 DNA 2,4 -BNA prefers A-type duplex 2,4 -BNA O4 -endo Energy C3 -endo C2 -endo B-type duplex C3 -endo locked RNA Stable as A-type duplex O4 -endo Energy C2 -endo A-type duplex Contribution to stability of 2,4 -BNA/RNA duplex C3 -endo K. A. Brameld et al. J. Am. Chem. Soc. 1999, 121,

16 2,4 -BNA is compatible with in vivo usage 1. High resistance to nucleases (in blood serum) DNA BNA, DNA chimera 2. Low toxicity (no febrile reaction of mice) Remaining nucleic acid duplex Time (min) BNA 3. Easy transfection with general transfection reagents Highly compatible with in vivo usage PS: Phosphorothioate DNA Highly resistant to nucleases C. Wahlestedt et al. Proc. Natl. Acad. Sci. USA, 2000, 97,

17 Further bridges of BNA 2,4 -BNA (LNA) Ethylene-BNA (ENA) N-methyl-BNA (BNA NC ) K. Morita et al. Bioorg. Med. Chem. Lett. 2002, 12, 73. S. M. A. Rahman et al. Angew. Chem. Int. Ed. 2007, 46, RNA selectivity Good Good Very Good Nuclease resistance Good Very Good Very Good 17

18 Backbone modification #3: UNA- unlocked nucleic acid RNA UNA Flexible structure by lacking the C2 -C3 - bond of the ribose ring of RNA P. Nielsen et al. Bioorg. Med. Chem. Lett. 1995, 3, 19. Single-stranded RNA RNA duplex RNA/UNA duplex Unfavorable enthalpy loss reduction of duplex stability 18

19 UNA is highly suitable for sirna 1. UNA has high nuclease resistance. Stability of natural sirna and 2, 4 -BNA (LNA) or UNA-modified sirna in mice: Natural RNA BNA/UNA M. B. Laursen et al. Mol. Biosyst. 2010, 6,

20 UNA is highly suitable for sirna 2. UNA-modified sirna can reduce off-targeting Off-targeting (genes) Natural sirna + UNA at the ends + UNA at 7 th position of antisense Natural RNA UNA N. Vaish et al. Nucleic Acids Res. 2011, 39,

21 Summary of backbone modified artificial nucleic acids Nuclease resistance PNA BNA UNA Very High High High Binding affinity Strong Strong Weak Reduction of off-targeting Good Good Very Good (Tunable) Transfection Difficult Easy Easy Compatibility with enzymes Poor Good Good Probe Antisense oligo sirna oligo : Very suitable : usable : not usable 21

22 Summary Nucleic Acids 1. Base modifications 2. Backbone modifications Tuning of base pairings -Stronger base pairs -Repulsion of modified bases Unique nucleic acids such as PNA probes BNA antisense oligos UNA sirna Practical usage for researches and therapeutics (As nucleic acid drugs and research tools) 22