Polysaccharide−Polynucleotide Complexes. 2. Complementary Polynucleotide Mimic Behavior of the Natural Polysaccharide Schizophyllan in the Macromolecular Complex with Single-Stranded RNA and DNA

Abstract

Schizophyllan is an extracellular polysaccharide consisting of a beta-1,3-D-glucan main chain and exists as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO). When the single chain of schizophyllan (s-SPG) was mixed with poly(C), poly(A), poly(dA), or poly(dT), they form a macromolecular complex. On the other hand, poly(G), poly(U), poly(I), poly(dG), and poly(dC) do not. This nucleotide specificity evidences that the hydrogen bonds are essential to form the complex, because the former nucleotides have an unoccupied hydrogen-bonding site and the latter ones use the hydrogen-bonding sites in the intramolecular aggregation (i.e., such as the G quartet for poly(G) and poly(dG) and the U hairpin for poly(U)). The hypochromic effect and the increment in the circular dichroism (CD) intensity are observed in accordance with the complex formation. These facts indicate that the base stacking is enhanced in the complex. The solvent-composition (DMSO/water) dependence demonstrates that the hydrophobic interaction is important to form the complex as well as the hydrogen-bonding interaction. With increasing temperature the complex dissociates cooperatively and the melting curve enables the thermodynamic parameters to be evaluated (delta H = -60 to 70 kcal mol-1 and delta S = -150 to 200 cal mol-1 K-1). These values are comparable with those for double helix DNA. Namely, the complex can be characterized by enhancement of the base stacking, cooperative dissociation, the similar thermodynamic parameters to DNA, and combination of the hydrogen-bonding and hydrophobic interactions to form the higher-order structure. These facts surprisingly coincide with characters of the double helix of DNA. In other words, the s-SPG molecule behaves as if it were a complementary polynucleotide chain for the corresponding polynucleotide. Furthermore, stoichiometric study suggested that the complex structure is a triple helix consisting of two s-SPG and one poly(C) or poly(A) chains.

Optimal sequence of antisense DNA to silence YB-1 in lung cancer by use of a novel polysaccharide drug delivery system

“…SPG forms a stoichiometric complex with particular homo nucleotides such as poly(C) or poly(dA) via combination of hydrogen bonding and hydrophobic interactions (9)(10)(11). On the basis of our previous studies, we attached dA 40 to the AS-ODN to enable complex formation with SPG and thus efficient gene silencing (13,14).…”

“…1) has a main chain comprising β-(1➝3)-D-glucan and one β-(1➝6)-D-glycosyl side chain that links to the main chain at every three glucose residues (8). We found that SPG forms a complex with single-stranded homo-polynucleotides and examined the fundamental properties of this complex (9)(10)(11). We recently started to apply this complex to therapeutic oligonucleotide delivery, including CpG DNA (12) and siRNA (13).…”

Delivery of Antisense Oligonucleotides to Nuclear Telomere RNA by Use of a Complex between Polysaccharide and Polynucleotide

“…The molar ratio of SPG repeating unit to ODN base number was always fixed at 1.5:1.0, which is an excess of SPG over the stoichiometric number. 13 The detail method of the complexation of ODN with SPG was described elsewhere. 12 Telomerase Assays.…”

“…We have found that a natural polysaccharide, called schizophyllan (SPG), can complex with single-stranded homo polynucleotides, without using cation/anion interactions. 12,13 Here, SPG consists of -(1!3)-D-glucan main chain and one -…”

“…The complexation occurs via a combination of the hydrogen bonding and hydrophobic interactions. [12][13][14] Subsequent studies have revealed that the SPG/ODN complexes reduce the hydrolytic degradation of the bound ODN and thus increase the antisense effect. 15,16 In addition, various functional groups to enhance the cellular uptake can be attached to SPG, including RGD and oligo arginine.…”