SYNTHESIS OF 15N-LABELED RNA FRAGMENTS

Project Details

Description

The long-term goals of this proposal are to develop and demonstrate the
synthetic strategies necessary for the preparation and utilization of 15N
labeled RNA fragments. Such site-specifically 15 N labeled molecules
have the potential to be invaluable probes of nucleic acid structure and
interactions. In fact, there are some types of information which may be
available only from 15N NMR of 15 N labeled molecules. At present, modem
high-field spectrometers with the necessary capabilities are generally
available, but the synthetic methodology necessary for preparation of the
15 N labeled RNA fragments is not. The first goal of this research is to develop synthetic routes to the 15N
labeled purine nucleosides [1-15N] adenosine, [6-15N]-adenosine,[3-15N]-
adenosine, [7-15N]-adenosine,[1-15N]-guanosine, [2-15N]-guanosine, [3-
15N]-guanosine, and [7-15N]-guanosine. The second goal is to develop
methodology which will allow conversion of these labeled monomers into
15N labeled RNA fragments, in sufficient quantity and of sufficient
purity for use in NMR studies. These syntheses will be carried out by
an H-phosphorate method that has been used successfully for synthesis of
15N labeled DNA fragments. A particular advantage of the H-phosphorate
method is that the monomers can be recovered and reused. Present methods
for protection of ribonucleosides, however, do not give high enough
yields of yr protected monomers to be applicable to valuable labeled
nucleosides. Thus, an important synthetic aim of this proposal is to
develop new synthetic procedures which will make it possible to obtain
high yields of protected monomers. Achievement of this goal will be
useful for less expensive monomers as well. Our procedures will be
useful for any researchers needing improved methods for RNA synthesis,
and will be particularly valuable where scale and economy are important. The final goal is to begin to use these 15N labeled RNA fragments to
probe RNA structure and interactions in order to develop a better
understanding of the principles of RNA folding, the specificity of RNA-
protein interactions, and the ability of RNA to function as an enzyme.
Specifically, the [1-15N], [2-15N], and [6-15N] labels will be used
primarily to monitor base-pairing, while the [7-15N] and [3-15N] labels
will be used to probes hydration in the major and minor grooves, as well
as other poorly understand interactions that occur in complex RNA
structures.
StatusFinished
Effective start/end date1/1/945/31/02

Funding

  • National Institutes of Health
  • National Institutes of Health: $196,143.00
  • National Institutes of Health: $343,870.00
  • National Institutes of Health: $183,935.00
  • National Institutes of Health: $243,479.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $224,181.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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