A chemical synthesis of LNA-2,6-diaminopurine riboside, and the influence of 2′-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides on the thermodynamic properties of 2′-O-methyl RNA/RNA heteroduplexes

Abstract

Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids. Derivatives of 2,6-diaminopurine riboside (D) are one type of modified nucleotide. The presence of an additional amino group at position 2 relative to adenine results in formation of a third hydrogen bond when interacting with uridine. New method for chemical synthesis of protected 3′-O-phosphoramidite of LNA-2,6-diaminopurine riboside is described. The derivatives of 2′-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides were used to prepare complete 2′-O-methyl RNA and LNA-2′-O-methyl RNA chimeric oligonucleotides to pair with RNA oligonucleotides. Thermodynamic stabilities of these duplexes demonstrated that replacement of a single internal 2′-O-methyladenosine with 2′-O-methyl-2,6-diaminopurine riboside (DM) or LNA-2,6-diaminopurine riboside (DL) increases the thermodynamic stability (ΔΔG°37) on average by 0.9 and 2.3 kcal/mol, respectively. Moreover, the results fit a nearest neighbor model for predicting duplex stability at 37°C. D-A and D-G but not D-C mismatches formed by DM or DL generally destabilize 2′-O-methyl RNA/RNA and LNA-2′-O-methyl RNA/RNA duplexes relative to the same type of mismatches formed by 2′-O-methyladenosine and LNA-adenosine, respectively. The enhanced thermodynamic stability of fully complementary duplexes and decreased thermodynamic stability of some mismatched duplexes are useful for many RNA studies, including those involving microarrays.

Secondary Structure of a Conserved Domain in the Intron of Influenza A NS1 mRNA

“…Square brackets indicate possible alternative binding sites (if 5 or 6 nucleotides of probe bind) or region of binding (if predicted duplex has less than 5 canonical base pairs). Data in italics represent probes that bind in buffer II, but not in buffer I;b- LNA nucleotides are marked with superscript L , D represents 2,6-diaminopurine, nucleotides without superscript are 2′-O-methyl-nucleotides;c- S –strong binding, M – medium binding; underline – binding site with no ambiguity;d- buffer composition is 300 mM KCl, 10 mM MgCl 2 , 10 mM Tris-HCl pH 7.0;e- buffer composition is 1 M KCl, 10 mM MgCl 2 , 10 mM Tris-HCl pH 7.0;f- calculated by RNAstructure 4.6 program (for 1 M NaCl, assuming no structure of target RNA and unmodified probe), values correspond to binding sites listed in the first column;g- calculated for 100 mM NaCl buffer according to published equation [37], [38].Probes listed underneath the double line have no perfect Watson-Crick match of the first five probe nucleotides to sequences in the target, but bind to target with at least one predicted GU wobble pair or in the case of site 114 have perfect complementarity to the target with only last five nucleotides of the probe. There are no complementary probes for sites: 129–131 and 137 on universal microarrays.…”

“…Isoenergetic microarrays were designed to have modified probes with similar free energies of binding to unstructured RNA [36]–[38]. A universal microarray with 861 different sequences, printed in triplicate, was used.…”

Isoenergetic Microarrays To Study the Structure and Interactions of DsrA and OxyS RNAs in Two- and Three-Component Complexes

“…Because the central fragment of DsrA RNA is A and U rich, LNA and 2′-O-methylated and 2,6-diaminopurine nucleotide residues were used to enhance the thermodynamic stability of the oligonucleotide d1/DsrA RNA hybrid (5, 31): the calculated free energy of the hybrid is −27.9 kcal/mol (2). In consequence, the binding of d1 to the complementary region of DsrA RNA may result in linearization of this fragment and structural rearrangements in other regions.…”

“…Modifications of oligonucleotides result in the increase of thermodynamic stabilities of formed hybrids (5, 31). The calculated free energies of hybrids were −21.6, −27.7 and −19.6 kcal/mol, respectively for o1, o2 and o3 (2).…”

“…Phosphoramidites of LNA 2,6-diaminopurine riboside and 2′-O-methyl-2,6-diaminopurine riboside were synthesized as described previously (31). …”