Recent Methods for Purification and Structure Determination of Oligonucleotides

Abstract

Aptamers are single-stranded DNA or RNA oligonucleotides that can interact with target molecules through specific three-dimensional structures. The excellent features, such as high specificity and affinity for target proteins, small size, chemical stability, low immunogenicity, facile chemical synthesis, versatility in structural design and engineering, and accessible for site-specific modifications with functional moieties, make aptamers attractive molecules in the fields of clinical diagnostics and biopharmaceutical therapeutics. However, difficulties in purification and structural identification of aptamers remain a major impediment to their broad clinical application. In this mini-review, we present the recently attractive developments regarding the purification and identification of aptamers. We also discuss the advantages, limitations, and prospects for the major methods applied in purifying and identifying aptamers, which could facilitate the application of aptamers.

Purely enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

“…Furthermore, we incorporated a 13 C/ 15 N-labeled nucleotide at the specific site, while other studies used either unlabeled or modified nucleotides (12,16,17,27). Our approach is similar enough to the published literature to expect a successful incorporation of the labeled nucleotide in the right position, as we have shown here.…”

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

“…These issues have been well characterized in the past, such as 5 -and 3 -inhomogeneity and low yield, due to suboptimal initiation sequences [19][20][21][22][23]. The full-length construct has to be purified from shorter and longer molecules, usually using polyacrylamide gel excision, ion-exchange or size-exclusion chromatography [24][25][26]. Issues with transcription have been addressed and solved to a large extent, but many of these solutions are not compatible with certain applications or do not increase the yield of difficult constructs.…”

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification

“…Therefore, longer alkyl chains in the ion-pairing buffer lead to longer retention times, while, on the contrary, if the fraction of organic modifier is increased, the analytes tend to leave earlier the column [122]. This technique is particularly suitable for the separation of RNA chains with only slight differences in the sequence and with length no larger than 60-mers.…”

“…This technique is particularly suitable for ONs modified with hydrophobic groups or with fluorescent dyes. The drawback is that this notoriously leads to the biomolecule denaturation, thus compromising its bioactivity[120] [122].…”