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Biomolecular NMR

Nucleic Acid Synthesis

Stable Isotope-Labeled Nucleic Acids and Related Compounds

  • Stable Isotope-Labeled Nucleic Acids and Related Compounds
  • Top 10 Reasons to Use Ammonium Salts
  • Example of RNA-Protein Interaction Studied by Multinuclear NMR
  • Testimonial from Dr. Michael Durney

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.

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Frequently Asked Questions 

Can buffers be added to the Cassia NTPs?

HEPES, Tris, or any other buffer can be added to the NTPs. The samples can then be refrozen or lyophilized, if needed. Care should be taken that adding the buffer (or any other adulterant) will not effect the downstream application.  

What is the pH of Cassia NTP solutions?

The Cassia NTPs are pH neutral, because of the ammonium salt counter-ion.

Can the Cassia NTPs be freeze-dried?

The NTP’s can be lyophilized if needed, however, there may be a small amount of hydrolysis that occurs during reconstitution.   Avoid freeze/thaw cycles.  All standard lab practices should be observed when lyophilizing and resuspending the material in solution.

Kevin Millis, PhD

Kevin Millis, PhD

Senior Scientist, Application Development Manager

Kevin Millis, PhD, is the Senior Scientist and Market Development Manager for all NMR and mass spectrometry product lines. Kevin is responsible for Technical Services both internally and externally for all CIL customers as well as being responsible for the application and market development for the CIL products.