Abstract Functional synthetic DNA or RNA ligands, known as aptamers, are a class of molecules with the versatility and tunable pharmacokinetics required for disease detection and therapy. The process by which aptamers are selected is called SELEX (Systematic Evolution of Ligands by EXponential enrichment). SELEX is a screening technology that allows the selection of oligonucleotide ligands against a variety of target molecules via an iterative and evolutionary process of continuous enrichment. A number of aptamer selection methods have become available to select aptamers against cell-surface proteins. Thus far, however, none of these methods has successfully identified aptamers specific to an antigen of a cell-surface receptor at its endogenous level and native conformation. Such precise targeting of known proteins is a determinant of successful diagnostic and therapeutic applications. Therefore, to select aptamers against the predetermined site of a cell-surface receptor in its native state, we, for the first time, introduced a simple method called Ligand-guided Selection (LIGS). Essentially, the iterative process in conventional SELEX is designed to winnow out low-affinity binders through a competitive process whereby high binders move on through the selection process. LIGS exploits this step by introducing a stronger, known high-affinity ligand against the target of interest to achieve two purposes: 1) directly outcompete and replace aptamers specific towards the target of interest and 2) introduce structural changes on the target protein upon binding of a secondary ligand to outcompete specific aptamers. Based on the specificity of a natural pre-existing ligand towards its target, the aptamers identified by LIGS are expected to show higher specificity towards the target ligand. This method is simple and adaptable to a number of platforms, including phage- display libraries and peptide libraries. Thus, the goal of this proposal is to further expand the LIGS technology and to develop aptamer-based immunotherapeutic molecules. To accomplish this, we will 1) identify a panel of aptamers against multiple surface markers from an evolved cell-SELEX pool against B-cell lymphoma, 2) identify multiple aptamers against overlapping regions of multidomain receptor sites of TCR-CD3 complex, and 3) develop an antibody mimetic by post-SELEX modification, followed by engineering dimeric aptamer constructs.