Effects of Locked Nucleic Acid Substitutions on the Stability of Oligonucleotide Hairpins

An understanding of the stability of nucleic acid folding is critical for applications involving RNA viruses, small molecule–RNA binding, and therapeutics, for example. To explore factors that affect this stability, hairpins made from oligonucleotides containing both a GAAA tetraloop and three to five complements in the stem have been used as models where locked nucleic acids (LNAs) have been substituted into the sequence. UV spectroscopy was used to obtain melting curves in 20% by volume formamide, and the enthalpies and entropies of melting were determined. Although LNA substitutions typically increase the stability of a hybrid, we have found a decrease in stability for DNA and RNA GAAA hairpins when LNA is substituted into the loop. Tetraloops synthesized from natural bases show higher enthalpies and entropies of melting compared to the LNA substituted sequences indicating that LNA substitutions can destabilize a hairpin but stabilize the corresponding double stranded structure.     

Aptamers—basic research, drug development, and clinical applications

Since its discovery in the early 1990s, aptamer technology has progressed tremendously. Automated selection procedures now allow rapid identification of DNA and RNA sequences that can target a broad range of extra- and intracellular proteins with nanomolar affinities and high specificities. The unique binding properties of nucleic acids, which are amenable to various modifications, make aptamers perfectly suitable for different areas of biotechnology. Moreover, the approval of an aptamer for vascular endothelial growth factor by the US Food and Drug Administration highlights the potential of aptamers for therapeutic applications. This review summarizes recent developments and demonstrates that aptamers are valuable tools for diagnostics, purification processes, target validation, drug discovery, and even therapeutic approaches.     

Increase in hybridization rates with oligodeoxyribonucleotides containing locked nucleic acids

Locked nucleic acids (LNAs) incorporated into either stable single stranded oligonucleotides containing tetraloops or their complements have been found to increase second order hybridization rate constants by an order of magnitude compared to the all-DNA hybridization rate constants. Model sequences composed of 20 bases in length that can form hairpins due to a stable GAAA tetraloop were used where LNAs were substituted for the nucleotides in the loop, stem, or end regions of the strand and in the complementary strand. Substitution of the LNAs to the loop predictably raised the melting temperatures of the duplex however, the hybridization rates between the tetraloop and the complementary sequence also increased. In contrast, when LNAs were substituted in the stem, the hybridization rate decreased implying the formation of a more stable hairpin. Substitution of LNAs into the end region of the sequence had little effect on the hybridization rate constants although melting temperatures still showed a predictable increase. Rates also increased when LNAs were substituted into complementary strands of DNA tetraloops. The increase in hybridization rate constant is being attributed to changes in the structure of the stable single strands.     

Fluorescent aptasensors based on conformational adaptability of abasic site-containing aptamers in combination with abasic site-binding ligands

Aptamers are nucleic acids that can selectively bind to a variety of targets. Aptamers usually undergo conformational transitions from a flexible or disordered structure into a rigid or ordered structure upon target-binding. This study describes a detection method for l-argininamide (l-Arm) and adenosine based on the conformational adaptability of nucleic acid aptamers. An abasic site (AP site) was formed in the stem and close to the target-binding site of a stem-loop aptamer as an anchoring pocket for a fluorescent ligand. 3,5-Diamino-6-chloro-2-pyrazine carbonitrile (DCPC), which can bind to AP site-containing DNA duplexes by pseudo-base pairing, was utilized as a signaling reporter for the target-binding. The binding of a target to an aptamer induces the tight pairing of bases flanking the AP site, so that DCPC can effectively bind to the stem. The binding of DCPC is accompanied by a significant enhancement of its fluorescence. This new sensing method without an antisense DNA strand was demonstrated by using l-Arm and its aptamer as a model. It was confirmed that the method can sensitively detect l-Arm with a detection limit of 2.1 μM. The proposed method was also applied to adenosine detection, where the reported sequence of an adenosine aptamer was slightly modified. The method based on an AP site-containing aptamer and an AP site-binding ligand was applicable to detection of a target in horse serum.     

Characterization and application of a novel RNA aptamer against the mouse prion protein

In order to isolate RNA aptamers against the mouse prion protein (mPrP), we carried out in vitro selection from RNA pools containing a 30-nucleotide randomized region. Aptamer 60-3 was found to have a high affinity for mPrP (K(d) = 5.6 +/- 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (K(d) = 22 +/- 4 nM). Following 5' biotinylation, aptamer 60-3 specifically detected PrP in mouse brain homogenate in a Northwestern blotting assay. To determine the mPrP-aptamer binding region, we performed protein-deletion-mutant analysis and competition-binding analysis using heparin. The results showed that aptamer 60-3 appears to have binding sites located between amino acids 23-108.     

Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease.

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DeltaNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DeltaNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DeltaNS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of DeltaNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DeltaNS3 with a Ki approximately 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.     

Determinants of apical loop-internal loop RNA-RNA interactions involving the HCV IRES

Domain II of the hepatitis C virus internal ribosome entry site is a major RNA structure involved in the viral mRNA translation. It comprises four different structural domains. We performed in vitro selection against the apical loop of the domain II and we identified RNA aptamers folding as an imperfect hairpin with an internal loop of interacting with the apical loop of the domain II. This RNA-RNA interaction creates apical loop-internal loop complex. The aptamer binds the target with an apparent K(d) of 35nM. In this study, the main structural elements of the target and the aptamer involved in the formation of the complex are characterized by mutation, deletion, and RNase probing analysis. We demonstrate that a complementary loop flanked by G,C rich upper and lower stems are crucial for such RNA-RNA interactions.     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

Application of locked nucleic acids to improve aptamer in vivo stability and targeting function

Aptamers are powerful candidates for molecular imaging applications due to a number of attractive features, including rapid blood clearance and tumor penetration. We carried out structure–activity relationship (SAR) studies with the Tenascin-C binding aptamer TTA1, which is a promising candidate for application in tumor imaging with radioisotopes. The aim was to improve its in vivo stability and target binding. We investigated the effect of thermal stabilization of the presumed non-binding double-stranded stem region on binding affinity and resistance against nucleolytic degradation. To achieve maximal thermal stem stabilization melting experiments with model hexanucleotide duplexes consisting of unmodified RNA, 2′-O-methyl RNA (2′-OMe), 2′-Fluoro RNA (2′-F) or Locked Nucleic Acids (LNAs) were initially carried out. Extremely high melting temperatures have been found for an LNA/LNA duplex. TTA1 derivatives with LNA and 2′-OMe modifications within the non-binding stem have subsequently been synthesized. Especially, the LNA-modified TTA1 derivative exhibited significant stem stabilization and markedly improved plasma stability while maintaining its binding affinity to the target. In addition, higher tumor uptake and longer blood retention was found in tumor-bearing nude mice. Thus, our strategy to introduce LNA modifications after the selection procedure is likely to be generally applicable to improve the in vivo stability of aptamers without compromising their binding properties.     

Conformational plasticity of RNA for target recognition as revealed by the 2.15?? crystal structure of a human IgG–aptamer complex

Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer–hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer–protein complexes. Moreover, the aptamer–hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.     

Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.     

Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure.

Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem. We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor. The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex. However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA. In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene. Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA. Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance.     

In vitro selection of RNA against kanamycin B.

Aminoglycosides are well-known antibiotics that function by interacting with ribosomal RNA in bacteria. In order to understand the molecular details between RNA and the drug, RNA aptamer was selected against kanamycin B. After 12 cycles of selection, RNA was cloned and sequenced. Among 9 clones, sequences of three clones were identical, suggesting the selected RNA was enriched. Among the cloned RNA molecules, the triplicated RNA was the maximum binding RNA. It showed a 180 nM affinity (KD) to the cognate aminoglycoside, as measured by a surface plasmon resonance, and a competition assay using a fluorescence anisotropy technique. The affinity of the maximum binding RNA to a similar aminoglycoside, tobramycin, was much stronger than 12 nM of KD. The binding site of the aminoglycoside in the maximum binding RNA was a stem loop located at the end of the 5' region. A stem loop structural motif, found in this study, was similar to those previously reported, even though the sequences of the RNA were totally different from the known sequences of the aminoglycoside binding site of other aptamers. The present study suggests that the aminoglycoside-binding region in RNA does not have a sequence specificity, but has a shape-specific bulged stem loop, even though it has a nanomolar affinity.     

In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

Structural basis for recognition of Co2+ by RNA aptamers

Co(2+) binding RNA aptamers were chosen as research models to reveal the structural basis underlying the recognition of Co(2+) by RNA, with the application of two distinct methods. Using the nucleotide analog interference mapping assay, we found strong interference effects after incorporation of the 7-deaza guanosine phosphorotioate analog into the RNA chain at equivalent positions G27 and G28 in aptamer no. 18 and G25 and G26 in aptamer no. 20. The results obtained by nucleotide analog interference mapping suggest that these guanine bases are crucial for the creation of Co(2+) binding sites and that they appear to be involved in the coordination of the ion to the exposed N7 atom of the tandem guanines. Additionally, most 7-deaza guanosine phosphorotioate and 7-deaza adenosine phosphorotioate interferences were located in the common motifs: loop E-like in aptamer no. 18 and kissing dimer in aptamer no. 20. We also found that purine-rich stretches containing guanines with the highest interference values were the targets for hybridization of 6-mers, which are members of the semi-random oligodeoxyribonucleotide library in both aptamers. It transpired that DNA oligomer directed RNase H digestions are sensitive to Co(2+) and, at an elevated metal ion concentration, the hybridization of oligomers to aptamer targets is inhibited, probably due to higher stability and complexity of the RNA structure.