Nucleic Acid Synthesis

NMR spectroscopy is an important tool to determine structures of nucleic acids alone and in interaction with proteins or small molecules. In fact, about half of the RNA structures deposited in the Protein Data Bank have been determined by NMR spectroscopy. Nucleic acids containing roughly less than ~40 nucleotides will require simple ¹⁵N or ¹³C/¹⁵N enrichment to provide the constraints necessary to determine full three-dimensional structures. Advances in isotope-enrichment strategies have been crucial in the success for the study of larger RNAs. These advances primarily include the use of selectively and uniformly deuterated nucleotides and segmental isotope labeling using deuterium. 

The most popular approaches to produce labeled RNA molecules for NMR studies use enzymatic in vitro transcription methods that employ labeled rNTPs, T7 RNA polymerase and either linearized plasmids or double stranded DNA as templates. These techniques are used to construct labeled RNA molecules of which all of one type of nucleotide is labeled.

Labeled DNA oligonucleotides are routinely synthesized using enzymatic in vitro methods that utilize labeled dNTPs, a DNA polymerase, and a cDNA template. One advantage of using enzymatic methods over phosphoramidite chemistry is that large oligonucleotides (e.g., >50 nucleotides in length) can be prepared in milligram quantities. Position-specific labeled DNA molecules can be synthesized using standard phosphoramidite chemistry (using CIL’s deoxyphosphoramidites) to overcome the limited chemical-shift dispersion of DNA, as well as to create residue-specific probes to obtain functional, structural, and dynamic information.

CIL's Partner for Over 15 Years:

 

Frequently Asked Questions