Analytical HPLC analysis of purified antisense oligonucleotide.

<p>Sample preparation: 25 µL purified antisense oligonucleotide; Column: DNAPac PA-100 (4/250); Flow rate: 1 ml/min; Buffer A: 10 mM NaClO₄+1 mM Tris; Buffer B: 300 mM NaClO₄+1 mM Tris; Gradient: 10–70% B, 7.6 CV.</p

Schematic description of PEAR.

<p>Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide (<i>X</i>) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos (<i>X/X′</i>) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.</p

Capillary electrophoresis of HPLC purified antisense oligonucleotide.

<p>Capillary: 50 µm×18 cm, filled with polyacrylamide cyclodextrin gel; Buffer: Tris-borate/Urea; Running conditions: 1.5 kV/30 min; Sample application: 1 kV/5 s.</p

Double digestion of the PEAR product by PspGI and Hpy99I.

<p>The sense and the antisense strand are indicated respectively by (+) and (<b>−</b>). Recognition sites for PspGI and Hpy99I are underlined and marked. Each position where cleavage is expected to occur is indicated by a caret (“<b> ̂</b>”). The antisense strands (<i>A</i>) are black boxed, the sense strands (<i>B</i> and <i>C</i>) are boxed, and the by-products (<i>D</i> and <i>E</i>) are grey boxed. The expected length for each strand is indicated in parenthesis.</p

ESI Mass spectrum analysis of HPLC purified antisense oligonucleotide.

<p>Calculated molecular weight is 6105.0 Daltons, the measured molecular weight is 6105.5 Daltons.</p

Average recoverable concentration, purity and yield of the purified antisense oligonucleotide.

a<p>The product of round 1 was used as seeds for round 2, and that of round 2 for round 3.</p>b<p>Each run consisted of 95×100 µl reactions. Target and probe concentration are at 1 nM and 100 nM respectively.</p>c<p>Round 1 had two duplicate runs. As the first run had been used as seeds, the second run was used for purification and analysis.</p>d<p>Purity was calculated as peak area % at UV 260 nm.</p

PEAR reactions with different target concentrations.

<p>Lane M: Invitrogen Trackit™ 10 bp DNA ladder; lane 2–8: PEAR reaction with 1 to 10⁻⁴ nM of input oligonucleotides. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Probe (X′R′X′R′X′) concentration is at 100 nM. A final incubation at 75°C for 30 min was conducted to cleave the product.</p

PEAR reactions with complete and incomplete components.

<p>Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.</p

HPLC separation and purification of antisense oligonucleotide.

<p>Fractions are indicated by letter A to E as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008430#pone-0008430-g004" target="_blank">Fig. 4</a>. Fraction A, which contains the antisense strands, was collected in the indicated interval. Sample preparation: 10 µg PEAR product double digested by PspGI and Hpy99I; Column: SOURCE Q PE 4.6/100; Flow rate: 1 ml/min; Buffer A: 10 mM NaOH, pH 12; Buffer B: 10 mM NaOH+2M NaCl, pH 12; Gradient: 20–35% B in 50 column volume.</p

Mutation of N406 in the NPPY motif abolishes MTase and cleavage activity.

<p>(A) Fluorescence spectra of WT and N406A RM.BpuSI. The high similarity of the two spectra indicated that mutation N406A does not induce significant conformation change to RM.BpuSI. Titration of SAM or SIN into mutant N406A does not show specific changes in fluorescence (data not shown). (B) Titration of SAM or SIN into WT RM.BpuSI in the presence of 400 μM ANS. The data points are fitted to a hyperbolic function and K_d values are found to be 76.4 and 71.1 μM for SAM and SIN, respectively. (C) Two-fold dilutions of WT or N406A RM.BpuSI was incubated with 1 μg of λ DNA in the presence or absence of 160 μM SAM at 37°C for 1 h. While WT exhibits enhanced cleavage activity in the presence of SAM, mutant N406A does not exhibit cleavage activity in the presence or absence of SAM.</p