Molecular assembly for high-performance bivalent nucleic acid inhibitor Youngmi Kim, Zehui Cao, and Weihong Tan* Center for Research at the Bio/nano Interface, Department of Chemistry, University of Florida Genetics Institute, Shands Cancer Center, and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200 Edited by Richard N. Zare, Stanford University, Stanford, CA, and approved February 7, 2008 (received for review December 14, 2007) It is theorized that multivalent interaction can result in better to a single-stranded DNA called the cellular retinol-binding affinity and selectivity than monovalent interaction in the design protein II element (CRBP-II element). Interestingly, although of high-performance ligands. Accordingly, biomolecular engineers the intrinsic affinity of one or more units of RXR-L for one are increasingly taking advantage of multivalent interactions to CRBP-II element (i.e., di-, tri-, or tetravalent interaction) is fabricate novel molecular assemblies, resulting in new functions insufficient to initiate transcription, more than five of these for ligands or enhanced performance of existing ligands. Substan- complexes adjacent to CRBP-II elements can, in fact, initiate the tial efforts have been expended in using small molecules or transcription. As a result, transcriptive response is well regulated epitopes of antibodies for designing multifunctional or better- depending on the concentration of the transcription factor. performing ligands. However, few attempts to use nucleic acid Furthermore, as noted above, this activity demonstrates the aptamers as functional domains have been reported. In this study, cooperative configuration, as noted above, which gives polyva- we explore the design of bivalent nucleic acid ligands by using lent interactions the potential for considerably increased binding thrombin and its aptamers as the model by which to evaluate its affinity. functions. By assembling two thrombin-binding aptamers with A number of recent studies have reported the unique prop- optimized design parameters, this assembly has resulted in the erties of multivalent interactions. Investigators have attempted successful development of a nucleic acid-based high-performance to mimic the mechanisms underlying such interactions to design bivalent protein inhibitor. Our experimentation proved (i) that the new therapeutic entities, particularly those using repetitive simultaneous binding of two aptamers after linkage achieved epitopes of antibodies (1). By designing more efficient targeting 16.6-fold better inhibition efficiency than binding of the monova- reagents with potentially viable therapeutic applications, all of lent ligand and (ii) that such an improvement originated from these attempts have shown promising results. A typical example changes in the kinetics of the binding interactions, with a koff rate is the single-chain variable fragment (scFv) constructed by Ϸ1/50 as fast. In addition, the newly generated aptamer assembly linking the antigen-binding VH and VL domains of an antibody is an excellent anticoagulant reagent when tested with different with a flexible polypeptide linker (4). The combinatory config- samples. Because this optimized ligand design offers a simple and urations of scFvs can be designed and investigated to optimize noninvasive means of accomplishing higher performance from the functionality. Another successful therapeutic design, which known functional aptamers, it holds promise as a potent anti- takes advantage of polyvalent interactions, is the bi-specific T thrombin agent in the treatment of various diseases related to cell engager molecule (BiTE) (5). A BiTE molecule is a bi- abnormal thrombin activities. specific antibody that is constructed by linking the binding domains of two antibodies with different specificities with short, anticogulation ͉ aptamers ͉ multivalent binding ͉ flexible peptides and is, therefore, expressed as a single polypep- thrombin tide chain. The typical working principle is that BiTEs bind with one arm to a target cell and the other arm to a T cell, n contrast to monovalent interaction, multivalent, or polyva- consequently activating the T cell. This unique mode of action Ilent, interactions involve the binding of multiple ligands of a results in increasing the cytotoxic potency of BiTE molecules at biological entity, such as small molecules, oligosaccharides, least 10,000-fold higher than that of conventional human IgG1 proteins, nucleic acids (NAs), lipids, or aggregates of these antibodies (6). These two achievements demonstrate how bio- molecules, to multiple binding pockets or receptors of a target, molecular engineers have exploited the potential of multivalent e.g., a protein, virus, bacterium, or cell (1). Polyvalency is binding motifs. However, the genetic engineering required to ubiquitous in biology and has a number of benefits over mono- mimic the mechanisms underlying multivalent interactions is valent interactions. For instance, polyvalent interactions collec- time consuming and prone to many technical difficulties. For tively possess higher binding affinity than the corresponding instance, expressed proteins may not fold into the expected monovalent interactions. That is, polyvalency results in a ‘‘co- tertiary structures, leading to nonfunctional products. Also, the operative’’ configuration in which the probability of rebinding of high molecular weight of the final product can be a limitation for a dissociated monomer to the target is increased by the presence future therapeutic applications. For these reasons, alternative of other monomers bound to the same target. A classical ligands that have functionality similar to that of antibodies, but example of this is demonstrated by the binding of galactose- without the limitations, are clearly attractive. In the present terminated oligosaccharides to C-type mammalian hepatic lec- tins (2). In addition to increased binding affinity, polyvalent Author contributions: Y.K. and Z.C contributed equally to this work; Y.K., Z.C., and W.T. interactions also stand a better chance of providing higher designed research; Y.K., Z.C., and W.T. performed research; Y.K. and Z.C. contributed new selectivity in target recognition. Noticeably, a multivalent reagents/analytic tools; Y.K., Z.C., and W.T. analyzed data; and Y.K., Z.C., and W.T. wrote binder, despite being composed of weak homo- or heteroge- the paper. neous ligands, can still have stronger binding property because The authors declare no conflict of interest. of multiple binding events. A well known example of this This article is a PNAS Direct Submission. phenomenon is taken from the biology of gene regulation by *To whom correspondence should be addressed. E-mail: [email protected]fl.edu. oligomeric transcription factors. Specifically, the retinoid X This article contains supporting information online at www.pnas.org/cgi/content/full/ receptor (RXR) functions as a transcription factor in the pres- 0711803105/DCSupplemental. ence of its ligand (3). Each RXR–ligand complex (RXR-L) binds © 2008 by The National Academy of Sciences of the USA 5664–5669 ͉ PNAS ͉ April 15, 2008 ͉ vol. 105 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711803105 Downloaded by guest on October 2, 2021 study, we demonstrate how the NA aptamer can be a strong candidate well suited for such multivalent applications. Aptamers are NA sequences selected by the Systematic Evo- lution of Ligands by Exponential Enrichment (SELEX) method (7, 8). They specifically recognize targets ranging from ions and small organic or inorganic molecules to proteins and living cells, with binding affinity and selectivity for targets comparable to those of antibodies. NA aptamers have markedly lower molec- ular weights (usually below 20,000) than antibodies, and their secondary structures are easily predictable. In addition, aptam- ers are not prone to the irreversible denaturation that affects antibodies. Moreover, they can be synthesized efficiently and reliably by using established phosphoramidite chemistry. Thus Fig. 1. Schematics of the working principles of monovalent and bivalent NA far, many aptamers have been identified, and some of them are ligands. (a) 15Apt, a monovalent ligand, has constant ON and OFF and diffuses very close to becoming marketable drugs (9–13). Nevertheless, into bulk solution immediately after dissociation from thrombin, resulting in engineering these aptamers for enhanced performance or new low inhibitory function. (b) In contrast, when linked to 27Apt to form a functions remains virtually unexplored. One of the most signif- bivalent ligand, 15Apt can rapidly return to the binding site after dissociation icant advantages of aptamers in molecular assembly for multi- because of molecular diffusion confined by 27Apt that is still bound to valent binding is the ease of conjugating aptamers together thrombin. As a result, the equilibrium of the reaction is shifted to the left side. through NA chemistry. This capability presents great potential in making aptamer conjugates with greatly enhanced functions or new and unique properties. explored to generate an enhanced functional NA ligand of Here, we demonstrate that rationally designed aptamer as- thrombin. However, this type of construct is likely to be even semblies can combine the functionality and binding affinity of more unpredictable, because its unique conformational struc- different aptamers to achieve greatly enhanced enzymatic inhi- ture can be disrupted, resulting in the loss of its binding property