Restriction endonucleases and T4 DNA ligase were from Thermo Fisher Scientific (Waltham, MA, USA). Oligonucleotides were synthesized in-house from commercially available phosphoramidites (Glen Research, Sterling, VA, USA). The sequences are listed in Table 2 . Oligonucleotides were 5′-labeled using γ[32P]-ATP (ICBFM Laboratory of Biotechnology, Novosibirsk, Russia) and T4 polynucleotide kinase (Biosan, Novosibirsk, Russia). Inhibitors were purchased from Vitas-M Chemical Ltd. (Hong Kong, China).
Phosphoramidites
Manufactured by Glen Research
Sourced in United States, Holy See (Vatican City State)
Phosphoramidites are key reagents used in the synthesis of synthetic oligonucleotides, which are short DNA or RNA molecules. They serve as building blocks for the automated, solid-phase synthesis of these oligonucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Regular oligonucleotides, by Sangon (3 mentions)
Oligonucleotides, by Glen Research (2 mentions)
Glen pak cartridges, by Glen Research (2 mentions)
Stains all dye, by Merck Group (2 mentions)
γ 32p atp, by PerkinElmer (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
T4 polynucleotide kinase, by Biosan (1 mentions)
Pu 2089plus, by Shiseido (1 mentions)
Uv 2075plus, by Shiseido (1 mentions)
Co 2067plus, by Shiseido (1 mentions)
Microtof q 2 instrument, by Bruker (1 mentions)
Fla 5100, by Fujifilm (1 mentions)
Capcell pak c18 mgii, by Shiseido (1 mentions)
Streptavidin c1 dynabeads, by Thermo Fisher Scientific (1 mentions)
Yeast trnaphe, by Merck Group (1 mentions)
Phage t4 polynucleotide kinase, by Biosan (1 mentions)
Molecular imager pharosfx system, by Bio-Rad (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
Hbs p buffer, by GE Healthcare (1 mentions)
38 protocols using phosphoramidites
1
Radiolabeling of oligonucleotides
2
Synthesis and Characterization of Modified DNA Oligonucleotides
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ODNs (Table 1 ) containing normal DNA bases and N1-methyladenine (m1A) and/or 2-aminopurine (2aPu) and FRET labels were synthesized on an ASM-700 Synthesizer (BIOSSET Ltd., Novosibirsk, Russia) using phosphoramidites purchased from Glen Research (Sterling, VA, USA) and were purified by anion exchange high-performance liquid chromatography. Concentrations of the ODNs were determined by measuring absorbance at 260 nm (A260). The purity, homogeneity, and integrity of each ODN were assessed by 20% polyacrylamide gel electrophoresis (PAGE) followed by Stains-All dye staining (Sigma-Aldrich, St. Louis, MO, USA). When needed, the modified DNA strands were 32P-labelled using [γ-32P]ATP and bacteriophage T4 polynucleotide kinase (SibEnzyme, Novosibirsk, Russia) and were purified by 20% denaturing PAGE.
Double-stranded substrate duplexes were prepared by annealing the modified strands (15m1A, 15m1A_2aPu, or 15m1A_FRET) and complementary strands (15T or 15TT in the case of a 2aPu-containing substrate) in a 1:1 molar ratio in a buffer consisting of 50 mM HEPES-KOH pH 7.5, 50 mM KCl, and 10 mM MgCl2. Single-stranded substrates have the ‘ss’ prefix, and double-stranded substrates the ‘ds’ prefix.
Double-stranded substrate duplexes were prepared by annealing the modified strands (15m1A, 15m1A_2aPu, or 15m1A_FRET) and complementary strands (15T or 15TT in the case of a 2aPu-containing substrate) in a 1:1 molar ratio in a buffer consisting of 50 mM HEPES-KOH pH 7.5, 50 mM KCl, and 10 mM MgCl2. Single-stranded substrates have the ‘ss’ prefix, and double-stranded substrates the ‘ds’ prefix.
3
Purification and Characterization of ssDNA
4
DNA and RNA Synthesis and Analysis
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The general chemicals were purchased from Wako Pure Chemical, Aldrich or the Tokyo Chemical Institute. The phosphoramidites and CPGs for the DNA synthesis were purchased from the Glen Research or ChemGenes Corporation. The target DNAs and RNAs were purchased from JBioS (Japan). The HPLC purification was performed by a JASCO HPLC System (PU-2089Plus, UV-2075Plus and CO-2067Plus) using a reverse-phase C18 column (CAPCELL PAK C18 MGII, Shiseido, 4.6 × 250 mm). MALDI-TOF MS measurements were performed by a Bruker MicrOTOFQ II instrument using a 3-hydroxypicolinic acid/diammonium hydrogen citrate matrix. The gel imaging and quantification were performed by a FLA-5100 (Fujifilm Co.).
5
Purification of DNA Repair Enzymes
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E. coli Fpg protein [40 (link)], human OGG1 protein [104 (link)], human POLκ (catalytic core, residues 1–560) [105 (link)], POLβ [106 (link)], POLλ [107 (link)], PrimPol [72 (link)], human DNA polymerase η (hPOLη) [72 (link)] and yeast DNA polymerase η (yPOLη) [108 (link)] were essentially overexpressed and purified as described. E. coli uracil–DNA glycosylase (Ung) was purchased from SibEnzyme (Novosibirsk, Russia) and trypsin was purchased from Samson-Med (St. Petersburg, Russia). Oligonucleotides (Table 2 ) were synthesized in-house from commercially available phosphoramidites (Glen Research, Sterling, VA, USA).
6
Synthesis and Characterization of Modified Oligonucleotides
7
In Vitro Transcription and Synthesis of Nucleic Acid Targets
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The polymerase ribozyme and all RNA templates were prepared by in vitro transcription, as described previously (14 (link)). Target DNAs were purchased from IDT. Most target RNAs were prepared by solid-phase synthesis employing phosphoramidites and other reagents from Glen Research (Sterling, VA, USA), with the exception of yeast tRNAPhe, which was purchased from Sigma-Aldrich (St. Louis, MO, USA). All modified NTPs and dNTPs were purchased from either Jena Bioscience (Jena, Germany) or TriLink BioTechnologies (San Diego, CA, USA). Streptavidin C1 Dynabeads were purchased from ThermoFisher (Grand Island, NY, USA).
8
Synthesis and Purification of Self-Complementary RNA
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An RNA oligomer containing a self-complementary region (5’-AAAAACGCGCGAAAAA-3’, 5181.31 Da) was designed and synthesized using standard β-cyanoethyl phosphoramidite chemistry and 2’-O-TBDMS-protected ribonucleosides. Phosphoramidites were purchased from Glen Research. Deprotection and initial purification of the RNA were carried out using Glen-Pak RNA purification columns. The oligonucleotide was further purified using polyacrylamide gel electrophoresis. The RNA was analyzed by MALDI-MS. See Supporting Data file for details.
9
Oligodeoxynucleotide Synthesis and Purification
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The oligodeoxynucleotides
(ODNs) were synthesized at the DNA/Peptide Core Facility at the University
of Utah using commercially available phosphoramidites (Glen Research).
The ODNs were then deprotected45 (link) and purified
via high-performance liquid chromatography (HPLC) following standard
protocols that are further explained in theSupporting
Information (Experimental and Figure S1) .
(ODNs) were synthesized at the DNA/Peptide Core Facility at the University
of Utah using commercially available phosphoramidites (Glen Research).
The ODNs were then deprotected45 (link) and purified
via high-performance liquid chromatography (HPLC) following standard
protocols that are further explained in the
Information
10
Synthesis and Characterization of Modified Oligonucleotides
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Oligonucleotides containing 5fC, 5mC, 5hmC or 5caC were synthesized using the ABI Expedite 8909 Nucleic Acid Synthesizer. The modified nucleotides were site-specifically incorporated at desired positions (Supplementary Table 1 ) with commercially available phosphoramidites (Glen Research). Subsequent deprotection and purification were carried out with Glen-Pak Cartridges (Glen Research) following the manufacturer’s instructions. Purified oligonucleotides were characterized by MALDI-TOF (< 40-mer). Regular oligonucleotides (and PCR primers) were purchased from Sangon Biotech.
Long duplex DNAs (Supplementary Table 1 and 2 ) were prepared through ligation of short duplexes (20–40 bp) with sticky overhangs21 (link). In brief, the ligation-site oligonucleotides were phosphorylated with T4 polynucleotide kinase (NEB) and then annealed with the corresponding complementary strands. Annealed duplexes with sticky overhangs were mixed and ligated with T7 DNA ligase (NEB) at 16 °C for 4 h, followed by purification with native PAGE (10%).
“10% 5fC” dsDNA and “5xC mix” dsDNA used in the qPCR assay were prepared through PCR amplification as previously described18 (link). All modified dCTPs were purchased from Trilink.
Long duplex DNAs (
“10% 5fC” dsDNA and “5xC mix” dsDNA used in the qPCR assay were prepared through PCR amplification as previously described18 (link). All modified dCTPs were purchased from Trilink.
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