SYNTHESIS, STRUCTURE, AND DEOXYRIBONUCLEIC-ACID SEQUENCING WITH A UNIVERSAL NUCLEOSIDE - 1-(2'-DEOXY-BETA-D-RIBOFURANOSYL)-3-NITROPYRROLE

A nucleoside analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrol e (4) was designed to function as a universal replacement for any of t he natural nucleosides in DNA sequences. Compound 4 was synthesized by the reaction of 3-nitropyrrole with sodium hydride and o-2-deoxy-3,5- di-O-toluoyl-D-erythropentofuranose, and the structure was confirmed b y X-ray diffraction. Nucleoside 4 was transformed to 3'-O-(2-cyanoethy l-N,N-diisopropylphosphoramidite) (6) for incorporation into oligonucl eotides by conventional synthesis protocols. Analogues of the oligonuc leotide, 5'-d(CGT AAT CAG AAA ACA AT)-3' with nucleoside 4 replacing t he natural nucleosides in UP to 9 positions were constructed and teste d as primers for dideoxy sequencing. Sequencing studies show that a su bstantial number of nucleotides can be replaced by 4 without loss of p rimer specificity. Sequencing primer 4 with substitutions of 4 at the third position in each of four codons gave a sequencing ladder compara ble to primer 1, the exact match, while a 256-fold degenerate oligonuc leotide mixture (primer 2) gave an unreadable sequencing ladder. Prime rs containing two or more mismatches gave indecipherable results. A un ique property of 4 was its ability to replace long strings of contiguo us nucleosides and still yield functional sequencing primers. Sequence s with three (primer 8), six (primer 9), and nine (primer 10) 4 substi tutions all gave readable sequencing ladders. Optical thermal profiles obtained for the oligonucleotide pairs 5'-d(C(2)T(5)XT(5)G(2))-3' and 5'-d(C(2)A(5)YA(5)G(2))-3' (X, Y = A, C, G, T, and 4) fit the normal sigmoidal pattern observed for the DNA duplex to single strand transit ion. The melting temperatures (T-m) of the oligonucleotides containing X-4 base pairs (X = A, C, G or T, Y = 4) all fell. within a 3 degrees C range of one another. However, the T-m's were significantly lower t han the corresponding sequences containing only A-T and GIC base pairs . The ability of 4 to associate by stacking with a natural nucleoside was confirmed by constructing the dimer d(Ap4) and determining the CD spectrum.