Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.

L.R Yarbrough,J.G Schlageck,M Baughman

doi:10.1016/s0021-9258(19)86429-3

Abstract

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.

Highlights

A new class of fluorescent nucleotide analogs which contain the fluorophore l-aminonaphthalene-&sulfonate attached via a y-phosphoamidate bond has been synthesized
Fluorescent Nucleotide Analogs communication we describe the synthesis of such analogs and their spectroscopic and biological properties
The (y-AmNS)NTPs should be excellent probes for the study of DNA-dependent RNA polymerases and perhaps other systems involving the utilization of nucleoside triphosphates

Summary

Introduction

A new class of fluorescent nucleotide analogs which contain the fluorophore l-aminonaphthalene-&sulfonate attached via a y-phosphoamidate bond has been synthesized Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. A variety of nucleotide analogs have been synthesized which have proven extremely useful in obtaining information about such processes These include those with altered phosphoryl structures such as AMP-PNP [1, 2] and ATP-y-S [3], analogs with modified ribose rings [4, 5], and others having altered purine or pyrimidine ring structures such as E-ATP [6] and S”-GTP [7].’.

Methods

Results

Conclusion