ABSTRACT
The novel bicyclic nucleoside (1S,5S,6S)-6-hydroxy-5-hydroxymethyl-1-(uracil-1-yl)-3,8-dioxabicyclo[3.2.1]octane [2'-deoxy-1'-C,4'-C-(2-oxapropano)uridine] (15), expected to be restricted into an O4'-endo furanose conformation, was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 5. The phosphoramidite derivative of 15 was successfully incorporated into oligodeoxynucleotides using standard methods, and thermal denaturation studies showed moderate decreases in duplex stabilities of -2.1 and -1.5 degrees C per modification toward complementary DNA and RNA, respectively.
Subject(s)
Bridged Bicyclo Compounds/chemistry , Nucleosides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , RNA/chemistry , RNA StabilityABSTRACT
The phosphoramidite (1S,3R,4S)-3-(2-cyanoethoxy(diisopropylamino)phosphinoxymethyl)-5-N-(4-monomethoxytrityl)-1-(uracil-1-yl)-5-aza-2-oxabicyclo[2.2.1]heptane 18 of a novel bicyclic nucleoside structure was synthesized from the known 1-(3'-deoxy-beta-D-psicofuranosyl)uracil 3. Conformational analysis of its structure verified its expected S-type furanose conformation, and the secondary amino group in the 4'-position allowed for incorporation into oligonucleotides using 5' --> 3' directed oligonucleotide synthesis as previously described for phosphoramidates. Thermal denaturation studies showed rather large decreases in duplex stabilities of -4.3 and -2.7 degrees C per modification toward complementary DNA and RNA, respectively.
Subject(s)
Bridged Bicyclo Compounds/chemical synthesis , Oligonucleotides/chemical synthesis , Uracil/chemical synthesis , DNA/chemistry , Indicators and Reagents , Nucleic Acid Conformation , Nucleic Acid Denaturation , RNA/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature , Uracil/analogs & derivativesABSTRACT
The phosphoramidite approach has been used for the automated synthesis of alpha-L-LNA, alpha-L-RNA, and oligomers composed of mixtures of alpha-L-LNA, alpha-L-RNA and DNA monomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. alpha-L-LNAs were shown to be significantly stabilized towards 3'-exonucleolytic degradation. Duplexes formed between RNA and alpha-L-LNA induced E. coli RNase H-mediated RNA cleavage, albeit very slow, at high enzyme concentration.
Subject(s)
Nucleic Acids/chemistry , RNA/chemistry , DNA/chemistry , Endoribonucleases/metabolism , Escherichia coli/enzymology , Exonucleases/metabolism , Nucleic Acid Hybridization , Nucleic Acids/metabolism , RNA/metabolism , StereoisomerismABSTRACT
To investigate the structural basis of the unique hybridization properties of LNA (locked nucleic acid) three novel LNA derivatives with modified carbohydrate parts were synthesized and evaluated with respect to duplex stabilities. The abasic LNA monomer (X(L), Figure 1) with the rigid carbohydrate moiety of LNA but no nucleobase attached showed no enhanced duplex stabilities compared to its more flexible abasic DNA counterpart (X, Figure 1). These results suggest that the exceptional hybridization properties of LNA primarily originate from improved intrastrand nucleobase stacking and not backbone preorganization. Two monocyclic seco-LNA derivatives, obtained by cleavage of the C1'-O4' bond of an LNA monomer or complete removal of the O4'-furanose oxygen atom (Z(L) and dZ(L), respectively, Figure 1), were compared to their acyclic DNA counterpart (Z, Figure 1). Even though they are more constrained than Z, the seco-LNA derivatives Z(L) and dZ(L) destabilize duplex formation even more than the flexible seco-DNA monomer Z.
