Oligo 2017 Oxford
Chemical ligation of DNA, biocompatibility and applications of artificial backbones
Tom Brown Click Chemistry: DNA strand ligation
Azides and alkynes can be easily attached to nucleic acids
They are unreactive towards the functional groups in nature; they react only with each other
The CuAAC click reaction works well in water
The CuAAC reaction can be switched on after annealing DNA strands by the addition of Cu I
The resultant triazole unit is extremely stable, and is not toxic Applications of artificial DNA linkages
The DNA ligase enzyme repairs single- stranded discontinuities in double stranded DNA molecules
DNA ligases have evolved to assist in DNA repair and DNA replication
Chemical ligation?
A. H. El-Sagheer and T. Brown, J. Amer. Chem. Soc. , 2009, 131, 3958-3964. Alternative to phosphodiester-triazole linkage
For chemically modified O B O DNA to have maximum utility it should behave like O normal DNA As in normal DNA PCR N Can a triazole linker be designed to resemble a N normal phosphodiester? N B O
As in normal DNA i.e. be tolerated by DNA polymerase enzymes? O triazole DNA template
4 Ligation of DNA Strands – Applications
Chemically modified gene synthesis e.g. epigenetics PCR amplification of triazole DNA is successful
Lane 1: DNA ladder
Lane 2: PCR product
peakdale .com PCR reads through triazole linkages correctly
TGTCC TGG TGCC GCG
also C-tz-T gives C-p-T and T-tz-T gives T-p-T
peakdale .com Rolling circle amplification
Reversed- Cyclisation phase mass HPLC of spectrum and RCA of cyclic 100- of cyclic 5’-azide-3’- mer 100-mer 3’-alkyne 100-mer
lanes 2 and 3; RCA of cyclic triazole ODN-31 and cyclic normal DNA using phi29 polymerase
El-Sagheer AH et. al. (2011) PNAS 108:11338–43. Linear copying of DNA (replication) and transcription to make RNA
Linear copying of 81-mer click- ligated triazole DNA template using DNA Polymerase I, Klenow Fragment Transcription of triazole DNA template to produce 60-mer RNA Lane 1: 18-mer 5’-fluorescein primer transcript using T7 RNA Polymerase Lane 2: copying of the triazole template Lane 1: reaction with unmodified DNA template Lane 3: copying of the normal template after 3 minutes Lane 2: reaction with triazole DNA template
Native and triazole DNA templates produced similar quantities El-Sagheer AH et. al. (2011) Chem Commun 47:12057-8. of RNA transcripts of the correct length Mass spec data on transcripts
Transcript Transcript from normal from triazole template template
El-Sagheer AH, Brown T. (2011) Chem Commun 47:12057-8. Is the triazole linker functional in vivo?
With Ali Tavassoli and Pia Sanzone Southampton University
Triazole linkages 80 bases apart
c Insert double stranded DNA into essential BLA gene that contains a triazole linkage in each strand Biocompatibility
Growth of colonies in repair-competent strain of E. coli 92%
96%
Growth of colonies in the UvrB deficient strain of E. coli JW0762-2
with Ali Tavassoli Southampton
El-Sagheer, A.H. et. al. Proc. Natl. Acad. Sci. USA 2011, 108(28): 11338–11343. Biocompatibility no selection pressure
Biocompatability of click DNA in E. coli.
(A) Left: transformants from amplification with native primers, Right: transformants of SDM with the triazole primers. 10 replicates, all colonies showing expected mCherry fluorescence.
(B) Comparison of the number of colonies in water (W), native (N), and triazole (T) plates. Triazole plates contain 93 ± 8 % of the colonies on the native plates, negative control contains < 1 %. Sanzone AP, et.al. (2012). Nucleic Acids Res, 40, 10567–10575 Biocompatibility no selection pressure
(A) and (B) Sequencing data of mCherry gene from colonies on triazole DNA plates. Forward strand in (A), complementary strand in (B), location of triazole backbone shown. (C) Lane 1 is DNA ladder, lane 2 BamHI attempted digestion of native pRSET mCherry plasmid, lane 3 is digestion of the progeny of the click-modified variant. The presence of the additional BamHI watermark incorporated by the click-SDM primers results in the highlighted 229 bp fragment in lane 3. Triazole linkage functional in human cells
With Ali Tavassoli and Pia Sanzone Southampton University
Human cells are shown to correctly transcribe through a non-natural, DNA backbone-linker.
-challenging the dogma that a phosphodiester backbone is essential for biological function of DNA
This is the first example of a non-natural DNA linkage being functional in human cells
Birts, C.N. et.al. (2014) Angew. Chem. Int. Edit. 53, 2362-2365. NMR structure of a DNA duplex with a triazole linkage
Normal DNA in orange, Triazole-containing DNA in blue, Triazole in green.
Triazole DNA has normal B-DNA Structure with some distortion at triazole linkage
With Andre Dallmann
Dallmann A, et.al. (2011) Chem. Eur. J., 17, 14714-14717 Structural basis for biocompatibility
Longer triazole backbone Normal DNA in orange, Triazole- has to allow base stacking, containing DNA in blue, Triazole in green. so 5’-carbon of deoxyribose is displaced.
Triazole N3 mimics phosphate oxygen
Dallmann A, et.al. (2011) Chem. Eur. J., 17, 14714-14717 Explanation of Biocompatibility Stability of duplexes containing triazole linkages
19 Stability of Triazole G-clamp duplex
Triazole G-clamp base paired with guanine Triazole stabilizes DNA:DNA and DNA:RNA duplexes
C CGACG CtC TGCAGC
(A). DNA:DNA duplexes; CD = control duplex, TD = triazole duplex, GTD = G clamp triazole duplex
(B). DNA:RNA hybrid duplexes; CD = control duplex, TD = triazole duplex, GTD = G clamp triazole duplex
>12 °C increase in Tm between triazole and G-clamp triazole G clamp triazole DNA is successfully ligated
… and the linkage is biocompatible
A. H. El-Sagheer and T. Brown, Chem. Sci., 2014, 5, 253-259. RNA click ligation: 100-mer Hairpin ribozyme Hairpin ribozyme synthesis by click chemistry
100-mer synthetic RNA labelled with 3 fluorescent dyes Confirmed by mass spec. Click reaction to synthesize hairpin ribozyme
Lanes 1-3: Starting hairpin arms,
Lanes 4-6: reaction mixture at different RNA
concentrations
N
N N Short hairpin ribozyme Calc. mass: 26983 Found mass: 26983
DNA-RNA hairpin ribozyme Calc. mass: 33170 Found mass: 33169.33 Hairpin ribozyme substrate cleavage
substrate
Lane 1: Substrate cleaved Lane 2: Substrate incubated in the presence substrate of the clicked hairpin ribozyme
El-Sagheer AH, Brown T. (2010) PNAS 107, 15329-15334 Third generation DNA backbone
Results on all triazole oligo T analogue of DNA (Isobe) suggest this backbone should give very stable duplexes Click ligation reaction
Click synthesis of 81-mer triazole DNA template Lane 1; click reaction mixture, lane 2; starting oligonucleotide (58-mer). PCR amplification
PCR of 81-mer triazole DNA template
Lane 1. 25 bp DNA ladder Control 61.59 deg C Lane 2. PCR reaction using 81-mer triazole DNA template Triazole 56.17 Lane 3. control primers without the template. Tm - 5.42 Alkylation of triazole
Shivalingam, A.; Tyburn, A. E. S.; El-Sagheer, A. H.; Brown, T., J. Amer. Chem. Soc. 2017, 139, 1575–1583. PCR kinetics
Hypothesis Replicating modified backbone is rate limiting in qPCR ∴ Longer extension time = more product
Taq Pol. 2 Phusion Pol. 2 TRUE FALSE
20 sec 20 sec 1.5 1.5 95 °C 95 °C T δ /
1 F
1 δ x sec 60 °C 0.5 0.5 Fluorescence (a.u.) Fluorescence (a.u.) 1 cycle 30 cycles 0 Ct 0 Ct 0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28 CycleCt CycleCt
Shivalingam, A.; Tyburn, A. E. S.; El-Sagheer, A. H.; Brown, T., J. Amer. Chem. Soc. 2017, 139, 1575–1583. Backbone steric demands
Slow
Smaller backbones replicated faster Fast Oligonucleotide Amide Ligation
=
Amide ligation of oligonucleotides is efficient and orthogonal to CuAAC chemistry Backbone steric demands
Phusion or Taq