Isolation of a nuclease-resistant decoy RNA that selectively blocks autoantibody binding to insulin receptors on human lymphocytes.

Abstract

SummaryAn RNA containing 2'-amino pyrimidines has been isolated using in vitro selection techniques that specifically and avidly (apparent Ka ,'-,30 nM) binds a mouse monoclonal antibody called MA20. This 2'-amino-derivatized RNA is at least 10,000-fold more stable than unmodified RNA in serum, and can act as a decoy and block MA20 binding to its natural antigen, the human insulin receptor, on lymphocytes. Furthermore, this RNA decoy can inhibit MA20-mediated downmodulation of insulin receptor expression on human lymphocytes in culture by up to 90%. Surprisingly, the decoy RNA cross-reacts with autoantibodies from patients with extreme insulin resistance and can inhibit these antiinsulin receptor antibodies from downmodulating insulin receptor expression by up to 80% without impeding insulin binding to its receptor. These results suggest that in vitro--selected decoy RNAs may be able to specifically and selectively block oligoclonal autoimmune responses to self-antigens in patients with autoimmune diseases.A utoimmune diseases are often caused by abnormal targeting of self-antigens by autoantibodies (1). A small "decoy" ligand could potentially interrupt this process if it specifically and avidly bound the autoantibodies and blocked them from interacting with their target antigen. A small peptide decoy seems the most obvious choice for such an inhibitor because a peptide should be able to structurally mimic the autoantigenic epitope on a self-protein. Unfortunately, isolation of small peptides that bind avidly to target proteins has been difficult, probably because short peptides cannot adopt stably folded structures very readily (2). By contrast, short RNA molecules can form fairly stable structures through basepairing interaction (3), and RNA decoys have been reported that avidly bind and block a variety of proteins (4-22). Expression of TAR and RRE decoy RNAs has been shown to render CD4 + T cells resistant to HIV replication by inhibiting the functions of the RNAbinding proteins tat and rev (8-12). Furthermore, by using in vitro selection techniques, "designer" decoy RNAs have been isolated from large pools of random RNA molecules that bind with high affinity and specificity to several proteins, including a few that do not naturally interact with nucleic acids (13,(15)(16)(17)(18)(19)(20)(21)(22).We previously identified a designer decoy RNA that binds and blocks the mAb, MA20 (18). This antibody naturally recognizes the main immunogenic epitope on the human insulin receptor (23) that elicits autoantibodies in patients with severe insulin resistance (type B) (24-26). A1-though this RNA was shown to be able to block MA20 binding to purified insulin receptors in solution in a test tube, we realized that the therapeutic utility of such a decoy RNA would be limited by its short half-hfe in the presence of serum nucleases. In addition, it was unclear if an RNA decoy made against MA20 could block an oligoclonal autoantibody response to the immunogenic epitope on the insulin receptor. Therefore, we sought to isolat...

Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

“…SE RNAs were internally radiolabeled and isolated as described Sullenger 1996, 1997;Bae et al 2002). Purified RNAs were incubated with proteins as described above.…”

“…In vitro selection was carried out essentially as described previously Sullenger 1996, 1997;Bae et al 2002), with a few modifications. A random pool of RNA oligonucleotides was produced by in vitro transcription of synthetic DNA templates with NTPs and T7 RNA polymerase.…”

Aptamers to Proteins

“…In vitro (12) 30 Doudna et al, 1995;Lee and Sullenger, 1996 Monoclonal antibody to acetylcholine receptor RNA In vitro (12 +12 more) 6-60 Lee and Sullenger, 1997;Seo and Lee, 2000;Hwang and Lee, 2002 Immunoglobulin E…”

“…In order to develop an RNA aptamer against MA20 that was nuclease resistant, a 2l-aminopyrimidine-modified RNA selection was conducted on MA20 (Lee and Sullenger, 1996). After 12 rounds of SELEX, a modified RNA aptamer was identified that bound to MA20 with a K d of 30 nmol/L.…”

“…To measure its capacity to resist nuclease degradation, unmodified RNA and modified RNA was incubated in 10% human serum and while the unmodified RNA degraded in 15 s, most of the amino-modified aptamer was still present as a full-length ligand after 24 h. The aptamer also blocked MA20 binding to the insulin receptor by 95% on human lymphocytes. Lee and Sullenger then showed that the modified MA20 aptamer could protect cells from MA20-dependent and autoantibody-dependent downregulation of the insulin receptor expression by up to 90% and 80% respectively (Lee and Sullenger, 1996).…”