Template-directed ligation on repetitive DNA sequences: a chemical method to probe the length of Huntington DNA

Several genomic disorders are caused by an excessive number of DNA triplet repeats.


Synthesis of PNA conjugates
The PNA oligomers, both the non-modified and the aminoethyllysine modified PNA oligomers, with Nterminal cysteine and C-terminal thioester moieties were synthesized on the solid phase by using Bocchemistry on Fmoc-Gly or Fmoc-Lys derivatized MBHA resin. The C-terminal thioester moiety was assembled by coupling of S(Trt)-mercaptopropionic acid on Fmoc-Gly derivatized MBHA resin, the trityl group was removed upon treatment with trifluoroacetic acid (TFA) and triisopropylsilane (TIS) (19:1, v:v), followed by coupling of Boc-beta-alanine. The linear PNA synthesis was carried out by using PyBOP (4 equiv.) and NMM (8 equiv.) as coupling reagent in DMF. The deprotection of Cbz-protecting group on the side-chain amine and exocyclic amine was achieved simultaneously during the final step of cleavage of the PNA oligomer from the resin using TFA, trifluoromethanesulfonic acid (TFMSA) and m-cresol as scavenger in the ratio 16:3:1. The crude product was precipitated in cold diethyl ether and cooling with dry ice. The precipitate was collected by decanting. After drying with argon the precipitate was dissolved in water ( 0.1 % TFA) and purified by reversed-phase (RP) HPLC. The final purity was checked by analytical RP-UPLC (C18

Melting temperature (T M ) measurements
The PNA-DNA duplexes (1:1 stoichiometry) were dissolved in 1 μM concentration in 10 mM NaH 2 PO 4 buffer pH 7.4 containing 150 mM NaCl. TCEP (20 mM) was added in case of cysteinyl-containing probes. Absorbance versus temperature profiles were obtained by monitoring at 260 nm by using a Varian Cary 100 UV spectrophotometer equipped with a peltier block, scanning from 95 °C to 20 °C with temperature increments of 0.5 °C per minute. The data (triplicates) were proceeded using OriginPro8 form OriginLab Corporation and T M values derived from the maximum of the derivative curves.

Kinetic measurements of templated reactions
Reactive PNA probes were dissolved in 10 μM stock solution in ligation buffer (10 mM NaH 2 PO 4 , 150 mM NaCl, 10 mM MesNa, pH adjusted to 7.4). Mercaptopropionic thioester were incubated for 3 h with mercaptoethanesulfonate (10 mM) prior to use. The cysteinyl-PNA was incubated for 10 min in ligation buffer prior to use. For kinetic measurements appropriate amounts of PNA probes (final concentration 1 μM) and template (final concentration 200 nM or 100 nM) were mixed and incubated at 50 °C (or other T if mentioned different). The solution was agitated by means of a thermoshaker. Samples were withdrawn at certain time and quenched with 1.3 % TFA and subsequently analyzed by UPLC and Maldi-Tof/MS. In case partial blocker DNA was used the template was incubated with partial blocker DNA prior in 1:1 stoichiometry for 30 min prior to addition of reactive probes. The yield of ligation product was determined by means of HPLC analysis. For kinetic analysis data was submitted to nonlinear Michaelis-Menten fitting.  The background reaction rate is high, even at 50 °C. Increasing the reaction temperature to 65 °C results in reduced background but at this temperature the thioester hydrolyzes rapidly (data not shown) and is therefore not available for native chemical ligation.

Polymerase chain reaction (PCR) experiments
The PCR was performed with an iQ5 real-time PCR from BioRad. Primers were designed with Primer3 (www.bioinfo.ut.ee/primer3) to have annealing temperatures around 60 °C (Nearest Neighbor method). The sequences of primers used in this study are for the left primer, biotine-GACCCTGGAAAAGCTGATGA and for the right primer, Alexa700-GGCTGAGGAAGCTGAGGAG to amplify only across the CAG-repeat stretch of the IT15 gene. The biotin-tag is used for streptavidin-based isolation of the amplified double strand DNA and the fluorescence dye is used for quantification of amplified single strand target DNA. The genomic Huntington Disease DNA (HD-DNA) was purchased from Coriell Cell Repositories, USA and the genomic wild-type DNA (WT-DNA) was purchased from Roche. The Taq-polymerase Kit was purchased from peqlab containing reactionbuffer Y (10×), enhancer solution P, MgCl 2 and Taq-polymerase (5 uμL -1 ). SYBRGold (10000×) was purchased by Invitrogen Molecular Probes and used in 10000-fold dilution.
The dNTP, containing all 4 nucleobases was purchased as a premixed Kit by peqlab. Due to the high GC content of the IT15 dGTP and dCTP (both peqlab, 25 μM) were added additionally.

Purification of dsDNA
The content of 40 wells was combined and the PCR product was separated from low molecular weight impurities by size exclusion with pre-packed spin columns from BioRad. The concentration of dsDNA was determined by means of Alexa Fluor 700 emission measured by using a NanoDrop ND-3300 Fluorospectrometer (excitation: 650 nm, emission: 723 nm). Typical yields of PCR reactions were 326 pmol (WT-DNA) and 269 pmol (HD-DNA).

Streptavidin binding procedure
Streptavidin-coated magnetic particles (20 mg, Roche) were washed twice with binding buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 7.5). dsDNA containing samples were diluted two-fold with binding buffer and added to the bead suspension. Suspensions were incubated for 2 h at room temperature with gently shaking. The beads were washed twice with washing buffer (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH 7.5). Subsequently, a NaOH solution (100 μL, 0.1 M) was added and the resulting suspension was incubated for 5 min. The suspension was neutralized with NaOAc (25 μL, 3 M) and the released ssDNA precipitated in cold EtOH. The supernatant was removed after 15 min spin down and the precipitated ssDNA disolved in H 2 O (30 μL). The ssDNA was quantified by fluorescence measurement of the Alexa Fluor 700 dye. Typical yields were 188 pmol for WT-DNA and 140 pmol for HD-DNA.