Aptamers for Targeted Delivery: Current Challenges and Future Opportunities

Aptamers are synthetic ssDNA/RNA molecules that are emerging as novel tools for the development of therapeutics, especially for targeted delivery. Aptamers are comparable to monoclonal antibodies, which are well-established therapeutic molecules, in terms of specificity and affinity to their target. The advantage of aptamers over antibodies includes their high stability, ease of synthesis, less batch-to-batch variation, easy chemical modifications that allow different conjugation chemistries, small size for better tissue penetration and low immunogenicity. These advantages make aptamers an important tool for use in therapeutics for targeted delivery. However, aptamers do have some limitations that have hindered their widespread clinical use as a therapeutic agent. Some of their common limitations include serum stability, renal filtration and endocytic escape. Other limitations that are more specific to aptamers include lack of diversity in the aptamer library, nuclease susceptibility and claims of aptamer specificity as well. This book chapter sheds light on these challenges, and using examples, it explains the scientific advancements that have been achieved in overcoming these limitations. We will end this chapter by discussing the use of high-throughput technology, which is the only way of truly industrializing the aptamer technology akin to the development of small molecule drugs

Application of aptamers in diagnostics, drug-delivery and imaging

Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their target with high specificity and affinity. Although aptamers are analogous to antibodies for a wide range of target recognition and variety of applications, they have significant advantages over antibodies. Since aptamers have recently emerged as a class of biomolecules with an application in a wide array of fields, we need to summarize the latest developments herein. In this review we will discuss about the latest developments in using aptamers in diagnostics, drug delivery and imaging. We begin with diagnostics, discussing the application of aptamers for the detection of infective agents itself, antigens/toxins (bacteria), biomarkers (cancer), or a combination. The ease of conjugation and labelling of aptamers makes them a potential tool for diagnostics. Also, due to the reduced off-target effects of aptamers, their use as a potential drug delivery tool is emerging rapidly. Hence, we discuss their use in targeted delivery in conjugation with siRNAs, nanoparticles, liposomes, drugs and antibodies. Finally, we discuss about the conjugation strategies applicable for RNA and DNA aptamers for imaging. Their stability and self-assembly after heating makes them superior over protein-based binding molecules in terms of labelling and conjugation strategies.Peer reviewe

Development of protein expression methods for structural and functional studies

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Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase).

Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1-313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies

K_D values and curve fitting details of B2, C35, C35_1, C35_2 and C35_3 using Bio layer interferometry.

<p>K_D values and curve fitting details of B2, C35, C35_1, C35_2 and C35_3 using Bio layer interferometry.</p

<i>In-silico</i> analysis of non-binding of C35.

<p>(a) LD2 crystal structure from PDB id: 1K05 (left) being compared with the LD2 structure in the side view and top view of C35 structure showing the masking of the hydrophobic binding region predicted through HMM based SAM-T08 software. The LD2 binding region and the masking regions are depicted by the bracketed region. (b) Docking control showing FAT (co-ordinates from PDB id: 1K05) and LD2 (co-ordinates from PDB id: 2L6F, NMR model # 1) interaction using Hex 6.3 software. (c) Docking of C35 with FAT showing non-interaction due to masking effect. The sidechains of the active residues are shown as red sticks. The hydrophobic patch—HP2 in FAT molecule, which preferentially binds to LD2 is shown as a space filling model in orange (part of helix 1 of FAT) and grey (part of helix 4 of FAT) colors.</p

Dissection of paxillin constructs (residues 1–313) followed by expression and interaction studies.

<p><b>(a)</b> Timeline for overall-strategy. <b>(b)</b> Illustration of solubility and activity level of linear dissected human paxillin (residues 1–313). <b>(c)</b> Phosphor screen image of filter assay for optimization of temperature for paxillin constructs (left). Tabular representation of paxillin constructs, negative and positive controls corresponding to each well in filter assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150153#pone.0150153.ref001" target="_blank">1</a>]. <b>(d)</b> Phosphor screen image of 10% SDS PAGE of ³⁵S labeled cell-free expressed samples after GST pull-down assay of the paxillin constructs A1–E1; The right panel shows fraction of interaction of each construct with respect to B2 (since B2 showed maximum level of interaction) <b>(e)</b> Illustration of solubility and activity of dissected C3 constructs. All experiments were performed in triplicates and averaged. To rule out non-specific interactions that might occur with GST tagged FAT, GFP that was expressed in cell-free system and a reaction without DNA template were used as negative controls.</p

Regulatory and masking regions around paxillin’s LD2 and LD4 and their circular dichroism spectra.

<p>(a) CD spectra of paxillin LD peptides (LD1-LD5) and constructs: B2, C3, C35 and D2. CD spectra of LD2, LD4, C35 and D2 constructs showed negative bands at 222nm and 206nm and a positive band at 192nm that confirms the presence of alpha helical content thus may behave as folded effector binding sites. However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites. (b) LD2 regulatory region (54–130) and masking region (167–224) evidenced by constructs B3, B4 and B5. (c) LD4 regulatory region (216–257) and masking region (280–313) evidenced by constructs D1, D2 and E1.</p

Tailor-made Ezrin actin binding domain to probe its interaction with actin in-vitro

Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well