RNA aptamers targeted for human αA-crystallin do not bind αB-crystallin, and spare the α-crystallin domain

Abstract

The molecular chaperones, α-crystallins, belong to the small heat shock protein (sHSP) family and prevent the aggregation and insolubilization of client proteins. Studies in vivo have shown that the chaperone activity of the α-crystallins is raised or lowered in various disease states. Therefore, the development of tools to control chaperone activity may provide avenues for therapeutic intervention, as well as enable a molecular understanding of chaperone function. The major human lens α-crystallins, αA- (HAA) and αB- (HAB), share 57% sequence identity and show similar activity towards some clients, but differing activities towards others. Notably, both crystallins contain the "α-crystallin domain" (ACD, the primary client binding site), like all other members of the sHSP family. Here we show that RNA aptamers selected for HAA, in vitro, exhibit specific affinity to HAA but do not bind HAB. Significantly, these aptamers also exclude the ACD. This study thus demonstrates that RNA aptamers against sHSPs can be designed that show high affinity and specificity - yet exclude the primary client binding region - thereby facilitating the development of RNA aptamer-based therapeutic intervention strategies.

Keywords: Aptamer; Cataract; Chaperone; Crystallin; Melittin; RNA; SELEX; Small heat shock proteins.

Figure 1. Isolation of RNA aptamers for HAA

A. Schematic diagram of the SELEX process: “Gx” represents an aptamer candidate pool at the x-th generation of selection and amplification. Number of sequences in the two samples are indicated. B. Modification of washing conditions: The PCR products of the G13 DNA pool are shown. Three different conditions were used to wash the binding mixture on the filter, as indicated. Markers are the 100-bp ladder standards. C. Sequence of aptamers for HAA: Lowercase letters, constant region sequences; uppercase letters, variable region sequences. The two 25-base constant regions are each covered by a 22-base primer in PCR amplification. The number of identical sequences isolated in the G22 pool is indicated.

Figure 2. Characterization of Aptamers to HAA

A. Specific, concentration-dependent binding of aptamers to HAA. Each aptamer was radiolabeled (~1 nM, asterisk). Three different concentrations of HAA were used in binding reactions: 1.5 µg, 3 µg, and 4.5 µg. HAB (4.5 µg) was used to demonstrate specificity. BSA (4.5µg) was used as a control. In all binding reactions 10 µg yeast RNA (~100 nt in length) was used as a competitor (~30 mM or 60,000-fold in excess compared to target aptamer). B. Competition between aptamers for binding to HAA: Each radiolabeled aptamer (~1 nM, asterisk) was mixed with 1500× unlabeled aptamer in the binding reactions in the presence of 4µg HAA. Labeled and unlabeled aptamers were mixed together before adding protein. In all binding reactions, 10 µg yeast RNA was included. C. Effect of melittin on the binding of aptamers: Radiolabeled AptAC-2 was used in all binding reactions (concentration ~0.5 nM). HAA concentration (all lanes except the left most one, 4 µg, ~18 µM). Melittin used at three different concentrations: 1.5 µM, 3 µM, and 4.5 µM. Three different orders of addition were tested as indicated. In all lanes 10 µg yeast RNA (~100 nt) was included as competitor.

Figure 2. Characterization of Aptamers to HAA

A. Specific, concentration-dependent binding of aptamers to HAA. Each aptamer was radiolabeled (~1 nM, asterisk). Three different concentrations of HAA were used in binding reactions: 1.5 µg, 3 µg, and 4.5 µg. HAB (4.5 µg) was used to demonstrate specificity. BSA (4.5µg) was used as a control. In all binding reactions 10 µg yeast RNA (~100 nt in length) was used as a competitor (~30 mM or 60,000-fold in excess compared to target aptamer). B. Competition between aptamers for binding to HAA: Each radiolabeled aptamer (~1 nM, asterisk) was mixed with 1500× unlabeled aptamer in the binding reactions in the presence of 4µg HAA. Labeled and unlabeled aptamers were mixed together before adding protein. In all binding reactions, 10 µg yeast RNA was included. C. Effect of melittin on the binding of aptamers: Radiolabeled AptAC-2 was used in all binding reactions (concentration ~0.5 nM). HAA concentration (all lanes except the left most one, 4 µg, ~18 µM). Melittin used at three different concentrations: 1.5 µM, 3 µM, and 4.5 µM. Three different orders of addition were tested as indicated. In all lanes 10 µg yeast RNA (~100 nt) was included as competitor.