Boric acid

The hearts were rinsed in phosphate-buffered saline (PBS) three times for 10 minutes and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) at 4 °C overnight. Next, the samples were placed in a 4% acrylamide solution along with 0.5% w/v of the photoinitiator 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044, Wako Chemicals USA), followed by overnight incubation at 4 °C and incubation at 37 °C for 2–3 hours to initiate polymerization of the acrylamide. Once polymerized, the tissues were rinsed with PBS and placed into a clearing solution comprised of 8% w/v sodium dodecyl sulfate (SDS, Sigma Aldrich, USA) and 1.25% w/v boric acid (Fischer, USA) (pH = 8.5). The samples were incubated at 37 °C until they were clear, followed by one day in 1x PBS to remove residual SDS. The hearts were incubated in RIMS until transparent. Additionally, RIMS was made of forty grams of Sigma D2158 (Histodenz) in 30 ml of 0.02 M PB with 0.1% tween-20 and 0.01% sodium azide, pH to 7.5 with NaOH.

All oligonucleotides were purchased from Biomers. The sequences are listed in Table 1, with the mismatches on the RCA template marked in bold. T4 DNA ligase and 10X ligation buffer were purchased from Thermo Fisher. Φ29 polymerase and 10X polymerase buffer were purchased from Biosearch Technologies. Tris and boric acid were purchased from Thermo Fisher. EDTA disodium dihydrate was purchased from Sigma. ROTI GelStain Red was purchased from Carl Roth.

Aluminium chloride (99%), ammonium bicarbonate (≥99.5%), L-aspartic acid disodium salt (≥98%), L-alanine (≥99.5), β-alanine (≥99%), calcium chloride dihydrate (≥99%), chloroform (100–200 ppm amylene stabiliser, ≥99.5), cobalt chloride hexahydrate (cell culture grade), glycine (≥99%), glutamate (≥99%), guanidine chloride solution (8 M), hydrochloric acid (≥37%), isobutyl chloroformate (98%), manganese sulfate (cell culture grade), oxaloacetic acid (≥97%), potassium chloride (99%), potassium phosphate monobasic (99.5–101.0%), pyridine (98%), pyridoxamine dihydrochloride (cell culture grade), pyridoxal hydrochloride (≥99%), L-serine (≥99%), sodium acetate (99–101%), sodium orthovanadate (99.98%), and zinc chloride x (≥98%) were purchased from Sigma. Boric acid (analytical grade), water (HPLC grade) and acetonitrile (HPLC grade) were purchased from Fischer. Copper sulfate hexahydrate (98.0–102.0%), isobutanol (99%), magnesium chloride hexahydrate (≥98%), and nickel chloride hexahydrate (98%) were from Alfa Aesar. Sodium hydroxide was purchased from VWR, and sodium chloride (research-grade) was purchased from Millipore. Ammonium chloride (analysis-grade) was from Acros and 9-fluroenylmethoxycarbonyl chloride (97%) was from Novabiochem. Helium (99.995%) and Nitrogen (99.998%) were purchased from BOC.

CA, tris, magnesium chloride hexahydrate (MgCl₂·6H₂O), sodium chloride (NaCl), glacial acetic acid, and urea were used as purchased from Sigma-Aldrich. Boric acid was obtained from Thermo Fisher Scientific and used as supplied. Acrylamide/bis-acrylamide (40% 19:1) solution, ammonium persulfate, and tetramethylethylenediamine were used as purchased from BioShop Canada Inc.
d(A₁₅) oligonucleotides were synthesized on a MerMade-12 synthesizer, purified by denaturing polyacrylamide gel electrophoresis (PAGE; 20%, 1× tris-borate EDTA running buffer, and 8 M urea) and desalted with Gel-Pak desalting columns from Glen Research. Purity of the strand was confirmed by high resolution mass spectrometry (calculated mass, 4635.18; observed mass, 4634.28).
Stock solutions of 20 mM CA were prepared by dissolution in 100 ml of Milli-Q water in a volumetric flask and adjusted with acetic acid to pH 4.5. To properly dissolve and degas the solutions, they were heated at 65°C and sonicated and then cooled down to room temperature before being used.
Samples of 100 μl of dA₁₅ (25 μM) and CA (7.5, 10.0, 12.5, and 15.0 mM) in pH 4.5 Mg(OAc)₂ buffer (7.6 mM) were made in quadruplicates. A thin layer (~30 μl) of silicon oil was applied on top to prevent evaporation during experiments.

The total nitrogen and protein content of plant tissues was determined using the Kjeldahl apparatus (Perkin Elmer, Analyst 4000, Waltsman, MA, USA) by following the methodology as specified previously by Saez-plaza [27 (link)]. The dried plant samples (100 mg) were digested with digestion mixture (10 mL) in a digestion assembly. Subsequently, 2 mL of concentrated sulfuric acid (H₂SO₄) was added in mixture and incubated until a clear solution was formed. Subsequently, the digested mixture was then transferred in the distillation assembly and 4 mL of 2% sodium hydroxide (w/v)) and 50 mL of 1% boric acid (Thermo Fisher Scientific, Bedford, MA, USA) was added. After that, 20 mL of distilled water (DW) was added to the solution. One drop at a time of methyl red was added to the sample, and the sample was titrated with H₂SO₄ before the normality level was reached. The total protein-nitrogen contents were determined by the given formula
$$\begin{array}{c}\mathrm{Total}\mathrm{nitrogen}\left(\mathrm{g}/\mathrm{g}\right)\hfill \\ =\frac{\mathrm{Volume}\mathrm{of}\mathrm{sample}-\mathrm{Volume}\mathrm{of}\mathrm{blank}\times 0.1\mathrm{N}}{\mathrm{Dry}\mathrm{weight}\mathrm{of}\mathrm{sample}}\hfill \\ \times 100\hfill \end{array}$$
Total protein (g/g) = Total nitrogen % × 6.25
(Protein factor = 6.25, Nitrogen factor = 1.4007)

Soluble starch (PN: S9765), starch from corn (PN: S4126), starch from potato (PN: S4251), starch from rice (PN: S7260), starch from wheat (PN: S5127, unmodified), I₂ (PN: 207772, ≥ 99.8%), 85% (w/w) o-phosphoric acid (PN: 79620), and acetic acid (PN: 695092, ≥ 99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
D-Glucose (PN: 8337) and KI (PN: 105043) were purchased from Merck (Darmstadt, Germany). Boric acid (PN: A79-212, ≥99.5%) and NaOH (PN: S/4920/60) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). 37% (w/w) HCl (PN: 131020) was purchased from Panreac (Barcelona, Spain). Glucose oxidase / peroxidase (GOPOD) (K-GLUC) assay kit was purchased from Megazyme (Wicklow, Ireland).
Some examples of amylolytic enzymes assessed by this method were: amyloglucosidase from Aspergillus niger (PN: A9913) (illustrative results with this enzyme will be shown in the next sections), and alpha-amylase from Bacillus licheniformis (PN: A3306, heat-stable), purchased from Sigma-Aldrich, amyloglucosidase Spirizyme® Achieve and alpha-amylase from B. licheniformis Liquozyme® SC DS (AA-2), supplied by Novozymes (Bagsvaerd, Denmark). Detailed information is available in our previous publications [7 ,8 (link)].