Understanding of the structure and dynamics of nucleic acids is critical to exploit their many biological functions. Synthetic RNAs have been employed as therapeutics and molecular sensors. NMR is a suitable method to study proteins and nucleic acids in solution, however, the study of nucleic acids by NMR is more challenging than for proteins due to their unfavorable spectral properties. Incorporation of isotope into specific region of RNA or DNA is required for obtaining structural information by NMR. Stable isotope labeling enhances experimental sensitivity and spectral resolution, thus, are used to overcome extensive spectral crowding and increased resonance linewidths. 13C and 15N labeled nucleic acids have been applied to investigate RNA secondary structure and global conformation as well as to study dynamics of DNA flexibility. Creative Biostructure provides several isotope-labeling strategies to obtain stable isotope labeled DNA and RNA for NMR experiments.
Strategies for isotope enrichment of nucleic acids
Chemical synthesis of oligonucleotides is a versatile method to incorporate specific stable isotopes into nucleic acids. It is practical and cost-efficient for small nucleic acids.
RNAs for NMR-based experiments are commonly prepared by in vitro transcription using T7 RNA polymerase (RNAP) enzymes; T4 RNA ligase can be used for isotope labeling of 3’ end of RNA; DNA polymerases synthesize new DNA strands using isotope labeled dNTPs.
Large quantities of nucleic acids are typically purified by denaturing polyacrylamide gel electrophoresis. This method can be laborious. Purification under denaturing condition requires desalting and refolding of the nucleic acids prior to structural studies.
Efficient and reliable isotope labeling of biomacromolecules, such as protein, DNA, and RNA, requires specialized experience. Supported by dozens of scientists with stable isotope labeling expertise, Creative Biostructure dedicates to help clients with customized production of stable isotope labeled nucleic acids for NMR experiment. Please feel free to consult with us through online inquiry.
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