RNA and DNA aptamers as potential tools to prevent cell adhesion in disease

Abstract

Recent research has shown that receptor-ligand interactions between surfaces of communicating cells are necessary prerequisites for cell proliferation, cell differentiation and immune defense. Cell-adhesion events have also been proposed for pathological conditions such as cancer growth, metastasis, and host-cell invasion by parasites such as Trypanosoma cruzi. RNA and DNA aptamers (aptus = Latin, fit) that have been selected from combinatorial nucleic acid libraries are capable of binding to cell-adhesion receptors leading to a halt in cellular processes induced by outside signals as a consequence of blockage of receptor-ligand interactions. We outline here a novel approach using RNA aptamers that bind to T. cruzi receptors and interrupt host-cell invasion in analogy to existing procedures of blocking selectin adhesion and function in vitro and in vivo. Key words The SELEX techniqueThe SELEX method (systematic evolution of ligands by exponential enrichment) (1,2) is an oligonucleotide-based combinatorial library approach that has been extensively used to isolate high-affinity ligands (called aptamers) for a wide variety of proteins and small molecules (3-7). The in vitro selection of RNA and DNA ligands against specific targets obeys the same rules as natural selection. For this purpose a partial randomized synthetic DNA template is constructed containing a random inner region that is flanked on both sides by constant sequences. The random sequence classically consists of 15 to 75 random positions where all four bases are incorporated with equal probabilities. This pool containing 10 12 to 10 15 different sequences can be either directly used for selection or first transcribed to RNA using T7 RNA polymerase. In this case a T7 promoter site needs to be placed on the 5' site of the DNA template. The random DNA/RNA pool is exposed to the protein target and the best fitting molecules in the selection pool are culled and amplified. The procedure will be repeated with increasing stringency until the previous random pool is purified to a few molecules with the desired binding properties. The final pool is cloned into a bacterial vector and individual colonies are sequenced. The previous random regions are aligned and searched for consensus motifs. Consensus motifs often located in stem-loop structures are thought to mediate binding specificity. These aptamers are suitable for applications based on molecular recognition of a target molecule including diagnostics and therapeutics. Recently, the

Amino Acid Metabolic Routes in Trypanosoma cruzi: Possible Therapeutic Targets Against Chagas; Disease

“…The experimental outline for the identification of nuclease-resistant RNA aptamers binding to cell adhesion receptors of T. cruzi has been reviewed [135]. Aptamers were developed that bind to the receptors of cell matrix molecules on the T. cruzi cell surface and recognize specifically the infectious trypomastigote form of the parasite [130].…”

Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules

“…Also, a new member of the T. cruzi 80-85-kDa glycoprotein family was shown to bind to LN (15). Together with parasite-binding sites for host thrombospondin, heparin sulfate and FN, this LN-binding site has been proposed to be a potential target for inhibition of invasion of host cells by T. cruzi by RNA aptamers (16,17). Taken together, these data led to the concept that specific interactions between T. cruzi and host ECM components play a pivotal role in parasite dissemination, mediating basement membrane and ECM degradation as well as adhesion to and invasion of host cells.…”

Biocombinatorial Selection of Carbohydrate Binding Agents of Therapeutic Significance

“…Heparan sulfate, for example, was implicated as a critical factor in mediating binding of herpes simplex virus (HSV-1) to the cell surface [77]. Aptamers capable of binding heparan sulfate polysaccharide identified in [57] may be able to block the infection.…”

“…In 2001 Ulrich et al [57] devised a clever selection scheme for aptamers that bind to the receptors of extracellular cell matrix (ECM) of T. Cruzi in its invasive (trypomastigote) stage. In this stage the host's extracellular cell matrix (ECM) molecules bind to the parasite surface to effect the host cell invasion.…”