Automated Purification and Quantification of Oligonucleotides

Ivanetich, Kathryn M.; Reid, Ralph; Ellison, Ross; Perry, Kathy M.; Taylor, Richard G.; Reschenberg, M.; Mainieri, Avantika; Zhu, Dongmei; Argo, J.; Cass, Darrell L.; Strickland, Corey

doi:10.2144/99274rr04

Cited by 6 publications

(4 citation statements)

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“…Oligonucleotides were prepared at the UCSF Biomolecular Resource Center and purified as described (15). [5-3 H]UTP (20 Ci/mmol) was from Moravec Biochemicals Inc.…”

Section: Methodsmentioning

confidence: 99%

Molecular Recognition of tRNA by tRNA Pseudouridine 55 Synthase

Ivanetich

et al. 1998

Biochemistry

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Escherichia coli tRNA pseudouridine 55 synthase catalyzes pseudouridine formation at U55 in tRNA. A 17 base oligoribonucleotide analog of the T-arm was equivalent to intact native tRNA as a substrate for pseudouridine 55 synthase, viz., the features for substrate recognition by this enzyme are completely contained within the T-arm. The structures and activities of mutant tRNAs and T-arms were used to analyze substrate recognition by pseudouridine 55 synthase. The 17-mer T-arm was an excellent substrate for the synthase, while disruption of the stem structure of the 17-mer T-arm eliminated activity. Kinetic data on tRNA mutants lacking single T-stem base pairs indicated that only the 53:61 base pair, which maintains the 7 base loop size, was essential for activity. The identities of individual bases in the stem were unimportant provided base pairing was intact. A major function of the T-stem appears to be the maintainence of a stable stem-loop structure and proper presentation of the T-loop to pseudouridine 55 synthase. The 7 base T-loop could be expanded or contracted by 1 base and still retain activity, albeit with a 30-fold reduction in kcat. Kinetic analysis of T-loop mutants revealed the requirement for U54, U55, and A58, and a preference for C over U at position 56. Base substitutions at loop nonconserved position 59 or semiconserved positions 57 or 60 were well tolerated. Comparison of pseudouridine 55 synthase and tRNA (m5U54)-methyltransferase revealed that both enzymes required the stem-loop structure. However, pseudouridine 55 synthase was not stringent for a 7 base loop and recognized a consensus base sequence within the T-loop, while tRNA (m5U54)-methyltransferase recognized the secondary structure of the 7 member T-loop with only a specific requirement for U54, the T-loop substrate site. We conclude that recognition of tRNA by pseudouridine 55 synthase resides in the conformation of the T-arm plus four specific bases of the loop.

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“…Oligonucleotides were prepared at the UCSF Biomolecular Resource Center and purified as described (15). [5-3 H]UTP (20 Ci/mmol) was from Moravec Biochemicals Inc.…”

Section: Methodsmentioning

confidence: 99%

Molecular Recognition of tRNA by tRNA Pseudouridine 55 Synthase

Ivanetich

et al. 1998

Biochemistry

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“…between 1996 and 2009 reporting the isolation of both hydrophobic and hydrophilic metabolites using adsorptive resins is included in the ESI. † Selected examples reporting the use of Amberchrom resins [334][335][336][337] for the isolation of secondary metabolites include glycosylated macrolactones with antihelmintic activity, such as 6-desmethyl-N-methyluvirucin A 1 (93) from cultures of the actinobacterium Nonomuraea turkmeniaca, 338 the antifungal virgineone (94) isolated from the saprotrophic fungus Lachnum virgineum, 339 and lucensimycins D-G (e.g. lucensimycin G, 95) from cultures of Streptomyces lucensis MA7349.…”

Section: Reviewmentioning

confidence: 99%

The isolation of water-soluble natural products – challenges, strategies and perspectives

et al. 2022

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“…To meet this demand, high-throughput and large-scale ODN production is required. Although significant progress has been made on high-throughput and large-scale ODN synthesis in the past few years, limited progress has been made on high-throughput and large-scale purification. − Currently ODN purification technologies mainly include HPLC and gel electrophoresis. Although HPLC has been adapted for large-scale purification, it is highly expensive, requires expensive instrumentation and large volumes of harmful organic solvents, and is not suitable for high-throughput purification.…”

Section: Introductionmentioning

confidence: 99%

Parallel, Large-Scale, and Long Synthetic Oligodeoxynucleotide Purification Using the Catching Full-Length Sequence by Polymerization Technique

Eriyagama

Shahsavari

Halami

et al. 2018

Org. Process Res. Dev.

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The catching by polymerization synthetic oligodeoxynucleotide (ODN) purification technique was shown to be potentially suitable for high throughput purification by purifying 12 ODNs simultaneously, to be convenient for large-scale purification by purifying at 60 μmol synthesis scale, and to be highly powerful for long ODN purification by purifying ODNs as long as 303-mer. LC-MS analysis indicated that the ODNs purified with the technique have excellent purity.

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Automated Purification and Quantification of Oligonucleotides

Cited by 6 publications

References 0 publications

Molecular Recognition of tRNA by tRNA Pseudouridine 55 Synthase

Molecular Recognition of tRNA by tRNA Pseudouridine 55 Synthase

The isolation of water-soluble natural products – challenges, strategies and perspectives

Parallel, Large-Scale, and Long Synthetic Oligodeoxynucleotide Purification Using the Catching Full-Length Sequence by Polymerization Technique

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