Antiproliferative Activity of G-rich Oligonucleotides Correlates with Protein Binding

Abstract

Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotidebinding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.

Inhibition of DNA Replication and Induction of S Phase Cell Cycle Arrest by G-rich Oligonucleotides

“…Therefore, we investigated the effects of GRO29A on the replication-associated proteins most likely to be modulated. These were the SV40 large T antigen, which is known to bind to and unwind G-quartet structures (42), and RPA, which is known to bind to nucleolin (20), the putative GRO-binding protein (9). Although we could observe the interaction between nucleolin and RPA, we found that this was not significantly affected by the presence of GROs.…”

“…This assay was then used to examine a series of GROs with a variety of antiproliferative activities. These oligonucleotides have been described previously (9), with the exception of GRO26B, which is an active analog of GRO29A that lacks the three 5Ј-thymidines. Fig.…”

“…Nucleolin has been identified by us as a GRO-binding protein (9), and by several other groups (58 -61) as a G-quartet-binding protein. Although the results in this paper do not clearly define a link between the molecular effects of GROs and their binding to nucleolin, they point to an effect by inhibition of helicase activity and DNA replication.…”

“…In addition, nucleolin has been implicated, directly or indirectly, in other roles including nuclear matrix structure (19), DNA replication (20), cytokinesis and nuclear division (21), and as a nucleic acid helicase (12,22). There have been numerous reports describing the presence of nucleolin in the plasma membrane of cells (9,(23)(24)(25)(26)(27)(28), suggesting a further * This work was supported by the Department of Defense Prostate Cancer Initiative, the National Institutes of Health, and the Commonwealth of Kentucky Research Challenge Trust. The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…These aptameric oligonucleotides often contain secondary structure elements such as hairpins or G-quartets. The formation of G-quartet structures is also thought to contribute to non-antisense growth inhibitory effects of G-rich phosphodiester and phosphorothioate oligonucleotides (7)(8)(9).…”