Dna and rna oligonucleotides

Manufactured by Integrated DNA Technologies

Sourced in United States

DNA and RNA oligonucleotides are synthetic, short, single-stranded nucleic acid molecules. They are designed to serve as building blocks for various applications in molecular biology, genetics, and biotechnology.

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Oligonucleotides (424 citations) DNA oligonucleotides (166 citations) RNA oligonucleotides (34 citations) RNA/DNA oligonucleotides (2 citations)

17 protocols using dna and rna oligonucleotides

Purification of Oligonucleotides by Electrophoresis

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DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT) and purified by 12% denaturing polyacrylamide electrophoresis.

Pham P., Malik S., Mak C., Calabrese P.C., Roeder R.G, & Goodman M.F. (2019). AID–RNA polymerase II transcription-dependent deamination of IgV DNA. Nucleic Acids Research, 47(20), 10815-10829.

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Scaffold DNA:RNA Hybrid Assembly

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Synthetic DNA and RNA oligonucleotides were obtained from Integrated DNA Technologies (IDT). The nucleic acids were dissolved in RNase-free deionized water at a concentration of 1 mM. To assemble the scaffold, template DNA and RNA were mixed at a 1:1 ratio, annealed by incubation at 95°C for 2 min, 75°C for 2 min, 45°C for 5 min, and then decreasing the temperature by 5°C every 2 min until reaching 25°C. The annealed template DNA:RNA hybrid was stored at −20°C until use.

Hao Z., Epshtein V., Kim K.H., Proshkin S., Svetlov V., Kamarthapu V., Bharati B., Mironov A., Walz T, & Nudler E. (2020). Pre-Termination Transcription Complex: Structure and Function. Molecular cell, 81(2), 281-292.e8.

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DNA-RNA Hybrid Assembly Protocol

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Synthetic DNA and RNA oligonucleotides were obtained from Integrated DNA Technologies (IDT). The nucleic acids were dissolved in RNase-free deionized water at a concentration of 1 mM. To assemble the scaffold, template DNA and RNA were mixed at a 1:1 ratio, annealed by incubation at 95°C for 2 min, 75°C for 2 min, 45°C for 5 min, and then by decreasing the temperature by 5°C every 2 min until reaching 25°C. The annealed template DNA:RNA hybrid was stored at −20°C until use.

Hao Z., Gowder M., Proshkin S., Bharati B.K., Epshtein V., Svetlov V., Shamovsky I, & Nudler E. (2023). RNA Polymerase Drives Ribonucleotide Excision DNA Repair in E. coli. Cell, 186(11), 2425-2437.e21.

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Oligonucleotide and Streptavidin Preparation

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DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies. Unmodified DNA and RNA oligonucleotides and biotin-modified DNA oligonucleotides were rehydrated in 10 mM sodium cacodylate buffer, pH 6.5, to 500 μM stock before further diluting to working concentration with the same buffer. Cy5-modified RNA oligonucleotide was dissolved in 10 mM HEPES buffer, pH 7.0, to stock concentration at 200 μM. Oligonucleotide sequences are listed in Supplementary Table S1.
Streptavidin tetramer was purchased from Promega Corporation. Alexa Fluor 488-conjugated streptavidin was purchased from Thermo Fisher Scientific. The protein batch used for the fluorescence experiments described here contained four fluorophores conjugated to streptavidin. Proteins were rehydrated and diluted in 10 mM HEPES buffer, pH 7.0.

Chen S., Xing L., Zhang D., Monferrer A, & Hermann T. (2021). Nano-sandwich composite by kinetic trapping assembly from protein and nucleic acid. Nucleic Acids Research, 49(17), 10098-10105.

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Oligonucleotide-Based Biochemical Assay

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All DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT) (nucleic acid sequences are shown in Table S1). Fluorescent ATP was purchased from Jena Biosciences. GTP was purchased from Invitrogen. All the other chemicals were purchased from Thermo Fisher Scientific or Sigma-Aldrich.

Bhowmik D., Du M., Tian Y., Ma S., Wu J., Chen Z., Yin Q, & Zhu F. (2021). Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS. Nucleic Acids Research, 49(16), 9389-9403.

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Preparation of Radiolabeled HIV-1 Nucleic Acid Substrates

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DNA and RNA oligonucleotides were obtained from Integrated DNA Technologies (IDT) (Coralville, IA). T4 polynucleotide kinase, proteinase K, SUPERaseIN, and Gel Loading Buffer II were purchased from Applied Biosystems (now Life Technologies) (Foster City, CA). [γ-³³P]ATP, [α-³³P]dCTP, and [α-³³P]GTP were purchased from PerkinElmer (Shelton, CT). Purified tRNA^Lys3 from human placenta was obtained from Bio S&T (Lachine, Québec, Canada). RNA transcripts were synthesized by using the Ambion MEGAshortscript T7 kit (Life Technologies). HIV-1 RT was obtained from Worthington (Lakewood, NJ). The SP2 peptide (FLGKIWPSHKGRPGNF) was purchased from GenScript (Piscataway, NJ). The sequences of the viral nucleic acid substrates and the RNA 200 and RNA 60 templates were derived from HIV-1 NL4-3 (GenBank Accession no. AF324493) (Adachi et al., 1986 (link)). The RNA 105 template was derived from the HIV-1 HXB2 sequence (GenBank Accession no. K03455), as described previously (Hargittai et al., 2001 (link)).

Wu T., Gorelick R.J, & Levin J.G. (2014). Selection of fully processed HIV-1 nucleocapsid protein is required for optimal nucleic acid chaperone activity in reverse transcription. Virus research, 193, 52-64.

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Oligonucleotide Design for Sequence Evolution

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DNA and RNA oligonucleotides (listed in Supplementary Table S1) were from Integrated DNA Technologies and if necessary were purified, by denaturing PAGE (8 M urea, TBE). The 2 poly-T (,T4, T6) and 3 poly-A (T5, T7, T8) starting sequences consist of 69 nt, containing an either 39 nt poly-A or poly-T sequence flanked by different 15 nucleotide (nt) primer binding sites on both ends (Supplementary Table S1). The sequences were designed with nonoverlapping primer binding sites to avoid cross contamination from different evolution-selection experiments.

Wachowius F., Porebski B.T., Johnson C.M, & Holliger P. (2023). Emergence of ATP- and GTP-Binding Aptamers from Single RNA Sequences by Error-Prone Replication and Selection. ChemSystemsChem, 5(5), syst.202300006.

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Nucleoside Analysis by Reverse-Phase HPLC

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Unless otherwise noted, water was purified with the Milli-Q Direct Q3. DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies, with HPLC purification.
Nucleoside analysis was performed by reverse-phase high-performance liquid chromatography (HPLC, Agilent 1260 Infinity II) using a C₁₈ stationary phase (Phenomenex, Luna® 5 μm C₁₈(2) 100 Å, 250 × 4.6 mm) and an acetonitrile/100 mM triethylammonium acetate gradient. Oligonucleotide concentrations were determined by Qubit 4.0 Fluorometer (Thermo Fisher Scientific) using the dsDNA HS Assay Kit (Invitrogen, Q32851). High-throughput DNA sequencing samples were quantified using a Qubit 4 Fluorometer, prepared on an Ion Chef instrument and sequenced on an Ion Torrent GeneStudio S5 Plus using Ion 530 Chips.

Mahdavi-Amiri Y., Chung Kim Chung K, & Hili R. (2020). Single-nucleotide resolution of N6-adenine methylation sites in DNA and RNA by nitrite sequencing. Chemical Science, 12(2), 606-612.

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Synthesis and Characterization of Nucleic Acid Analogues

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2’F-araNTPs (faATP, faCTP, faGTP, faUTP) were obtained from Metkinen Chemistry (Kuusisto, Finland). TNA triphosphates (tATP, tCTP, tGTP, tTTP) were chemically synthesized following known procedures (20 (link),21 (link)). DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA), purified by denaturing polyacrylamide gel electrophoresis, and quantified by UV absorbance via NanoDrop (ThermoFisher, Waltham, MA). YM-3 microcentrifugal concentrators were purchased from EMD Millipore (Billerica, MA). The Monarch DNA gel extraction kit was purchased from New England Biolabs (Ipswich, MA). Thermococcus gorgonarius (Tgo) DNA polymerase (exo-), Kod-RS (22 (link)) and Kod-RSGA (23 (link)) TNA polymerases (exo-), and Bst-LF were expressed and purified from E. coli as previously described (24 ). Sequenase™ Version 2.0 DNA polymerase was purchased from ThermoFisher (Pudong, Shanghai).

Wang Y., Liu X., Shehabat M., Chim N, & Chaput J.C. (2021). Transliteration of synthetic genetic enzymes. Nucleic Acids Research, 49(20), 11438-11446.

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Oligonucleotide Purification and Radiolabeling

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DNA and RNA oligonucleotides were purchased from Integrated DNA Technologies (IDT;
Coralville, IA) and purified by 15% denaturing polyacrylamide gel electrophoresis (PAGE) before use. [g-32 P]ATP, [a-32 P]CTP and [a-32 P]GTP were obtained from PerkinElmer Life Sciences; rNTPs, from Promega (Madison, WI, USA); and 3′deoxy ATP (3′dATP) and CMPCPP, from Jena Bioscience. H2O2 30% (w/w) solution and standard buffers and reagents were from Sigma-Aldrich or Thermo-Fisher.

Bao Y., & Landick R. (2021). Obligate movements of an active site-linked surface domain control RNA polymerase elongation and pausing via a Phe-pocket anchor.

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