Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

Purification of prostate-specific membrane antigen using conformational epitope-specific antibody-affinity chromatography

Prostate-specific membrane antigen (PSMA) is a type II membrane protein that has attracted significant attention as a target for immunioscintigraphic and radioimmunotherapeutic applications for prostate cancer. However, definitive studies on its substrate and inhibitor specificity as well as protein-protein interactions have been somewhat limited by difficulties in the purification of native PSMA. In this study, we optimized the purification of native PSMA from LNCaP cells using conformational epitope-specific antibody-affinity chromatography. Western blot analysis and an HPLC-based enzymatic activity assay were used to compare the yield and activity of PSMA purified by different methods. The ratio of purified PSMA in a native and active conformation was determined by quantifying the amount of non-native PSMA not retained in a second antibody-affinity isolation. The addition of both a neutralization step and the inclusion of Zn(2+) to the equilibration buffer in desalting step provides considerable enhancement in the yield of active PSMA from LNCaP cells.     

Selection of RNA aptamers specific to active prostate-specific antigen

A counter-SELEX procedure with recombinant purified active prostate specific antigen (PSA) was used to identify specific RNA aptamers against the active PSA. We developed two different kinds of counter-SELEX methods; one includes pre-clearance step with inactive proPSA protein, and the other with tagged GST protein. After 9 iterative selection cycles, several identical RNA aptamers can be identified from both counter-SELEX methods. Real-time PCR analysis and gel retardation experiment showed that the aptamers have a specific binding activity against the active PSA, but not for GST or proPSA. These aptamers could be of potential use as specific diagnostic, imaging and/or therapeutic agents against prostate cancer.     

Substrate specificity of prostate-specific membrane antigen

A series of putative dipeptide substrates of prostate-specific membrane antigen (PSMA) was prepared that explored alpha- and beta/gamma-linked acidic residues at the P1 position and various chromophores at the P2 position, while keeping the P1' residue constant as L-Glu. Four chromophores were examined, including 4-phenylazobenzoyl, 1-pyrenebutyryl, 9-anthracenylcarboxyl-gamma-aminobutyryl, and 4-nitrophenylbutyryl. When evaluating these chromophores, it was found that a substrate containing 4-phenylazobenzoyl at the P2 position was consumed most efficiently. Substitution at the P1 position with acidic residues showed that only gamma-linked L-Glu and D-Glu were recognized by the enzyme, with the former being more readily proteolyzed. Lastly, binding modes of endogenous substrates and our best synthetic substrate (4-phenylazobenzoyl-Glu-gamma-Glu) were proposed by computational docking studies into an X-ray crystal structure of the PSMA extracellular domain.     

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.     

Regression of hepatocarcinoma cells using RNA aptamer specific to alpha-fetoprotein

Alpha-fetoprotein (AFP) is a cancer-associated fetal protein and has long been utilized as a serum fetal defect/tumor marker to monitor distress/disease progression. In addition, AFP is closely associated with the proliferation of hepatocellular carcinoma. Thus, direct targeting of AFP has been recommended for a therapeutic strategy against hepatocellular carcinoma. In this study, we developed and characterized an RNA aptamer that specifically bound to the alpha-fetoprotein using SELEX technology. The aptamer interacted with the AFP with a K(D) of ～33 nM. Importantly, the identified aptamer specifically and efficiently inhibited the AFP-mediated proliferation of hepatocarcinoma cells in a dose dependent manner. Moreover, the aptamer efficiently down-regulated AFP-induced expression of oncogenes in the cells. These results indicate that an AFP-specific RNA aptamer could be a useful therapeutic and diagnostic agent against AFP-related hepatocellular carcinoma.     

Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics

The mode of inhibition for phosphoramidate peptidomimetic inhibitors of prostate-specific membrane antigen was determined by inhibition reversibility experiments. The results revealed that these inhibitors can be classified into three types: pseudoirreversible (compounds 1-3), moderately reversible (compounds 4-9), and rapidly reversible inhibitors (compounds 10 and 11). Representative compounds from each class were further evaluated for their ability to induce cellular internalization of PSMA. Results from these experiments revealed that the pseudoirreversible inhibitor 1 induced the greatest PSMA internalization. The discovery of pseudoirreversible PSMA inhibitors is expected to provide a new avenue of investigation and therapeutic applications for prostate cancer and neurological disorders.     

Properties of an LNA-modified ricin RNA aptamer

'Locked nucleic acids' (LNAs) are sugar modified nucleic acids containing the 2'-O-4'C-methylene-β-D-ribofuranoses. The substitution of RNAs with LNAs leads to an enhanced thermostability. Aptamers are nucleic acids, which are selected for specific target binding from a large library pool by the 'SELEX' method. Introduction of modified nucleic acids into aptamers can improve their stability. The stem region of a ricin A chain RNA aptamer was substituted by locked nucleic acids. Different constructs of the LNA-substituted aptamers were examined for their thermostability, binding activity, folding and RNase sensitivity as compared to the natural RNA counterpart. The LNA-modified aptamers were active in target binding, while the loop regions and the adjacent stem nucleotides remained unsubstituted. The thermostability and RNase resistance of LNA substituted aptamers were enhanced as compared to the native RNA aptamer. This study supports the approach to substitute the aptamer stem region by LNAs and to leave the loop region unmodified, which is responsible for ligand binding. Thus, LNAs possess an encouraging potential for the development of new stabilized nucleic acids and will promote future diagnostic and therapeutic applications.     

Optimizing phenylethylphosphonamidates for the inhibition of prostate-specific membrane antigen

A series of eight N-2-phenylethylphosphonyl derivatives of glutamic acid was prepared to determine if the inhibitory potency of a phenylethylphosphonyl derivative of glutamic acid against prostate-specific membrane antigen (PSMA) could be improved through rational substitutions on the phenyl ring. The design of these eight analogs was based upon the Topliss batchwise approach. Of the inhibitors from the first generation, the 3,4-dichlorophenyl analog exhibited the greatest improvement over the lead compound which was an unsubstituted phenyl derivative, while the 4-methoxyphenyl analog was essentially void of inhibitory potency against PSMA in single-dose studies. From the potency ranking order of the first generation, the parameter most important to the pharmacophore was determined to be pi + sigma. Attempts to optimize further the potency of inhibitors by preparing a second generation of compounds did not result in structures with greater potency than that of the 3,4-dichlorophenyl analog from the first generation. Based upon K(i) values, the 3,4-dichlorophenyl analog represented a potency improvement of nearly one order of magnitude. These results confirm further the usefulness of the Topliss approach to analog development when large library synthesis cannot be achieved readily.     

Assignment methodology for larger RNA oligonucleotides: Application to an ATP-binding RNA aptamer

The use of uniform 13C,15N labeling in the NMR spectroscopic study of RNA structures hasgreatly facilitated the assignment process in small RNA oligonucleotides. For ribose spinsystem assignments, exploitation of these labels has followed previously developed methodsfor the study of proteins. However, for sequential assignment of the exchangeable andnonexchangeable protons of the nucleotides, it has been necessary to develop a variety of newNMR experiments. Even these are of limited utility in the unambiguous assignment of largerRNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei.These problems can largely be overcome by the additional use of base-type selectively13C,15N-labeled RNA in combination with a judicious use of related RNAs with basesubstitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNAaptamer in complex with AMP. Complete sequential 1H assignments, as well as the majorityof 13C and 15N assignments, were obtained.     

Phosphoramidate derivatives of hydroxysteroids as inhibitors of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a membrane-bound cell surface peptidase which is over-expressed in prostate cancer cells. The enzymatic activities of PSMA are understood but the role of the enzyme in prostate cancer remains conjectural. We previously confirmed the existence of a hydrophobic binding site remote from the enzyme's catalytic center. To explore the specificity and accommodation of this binding site, we prepared a series of six glutamate-containing phosphoramidate derivatives of various hydroxysteroids (1a-1f). The inhibitory potencies of the individual compounds of the series were comparable to a simple phenylalkyl analog (8), and in all cases IC50 values were sub-micromolar. Molecular docking was used to develop a binding model for these inhibitors and to understand their relative inhibitory potencies against PSMA.     

Solution structure of an informationally complex high-affinity RNA aptamer to GTP

Higher-affinity RNA aptamers to GTP are more informationally complex than lower-affinity aptamers. Analog binding studies have shown that the additional information needed to improve affinity does not specify more interactions with the ligand. In light of those observations, we would like to understand the structural characteristics that enable complex aptamers to bind their ligands with higher affinity. Here we present the solution structure of the 41-nt Class I GTP aptamer (K(d) = 75 nM) as determined by NMR. The backbone of the aptamer forms a reverse-S that shapes the binding pocket. The ligand nucleobase stacks between purine platforms and makes hydrogen bonds with the edge of another base. Interestingly, the local modes of interaction for the Class I aptamer and an RNA aptamer that binds ATP with a K(d) of 6 microM are very much alike. The aptamers exhibit nearly identical levels of binding specificity and fraction of ligand sequestered from the solvent (81%-85%). However, the GTP aptamer is more informationally complex (approximately 45 vs. 35 bits) and has a larger recognition bulge (15 vs. 12 nucleotides) with many more stabilizing base-base interactions. Because the aptamers have similar modes of ligand binding, we conclude that the stabilizing structural elements in the Class I aptamer are responsible for much of the difference in K(d). These results are consistent with the hypothesis that increasing the number of intra-RNA interactions, rather than adding specific contacts to the ligand, is the simplest way to improve binding affinity.     

Molecular recognition of cAMP by an RNA aptamer

Two classes of RNA aptamers that bind the second messenger adenosine 3',5'-cyclic monophosphate (cAMP; 1) were isolated from a random-sequence pool using in vitro selection. Class I and class II aptamers are formed by 33- and 31-nucleotide RNAs, respectively, and each is comprised of similar stem-loop and single-stranded structural elements. Class II aptamers, which dominate the final selected RNA population, require divalent cations for complex formation and display a dissociation constant (K(D)) for cAMP of approximately 10 microM. A representative class II aptamer exhibits substantial discrimination against 5'- and 3'-phosphorylated nucleosides such as ATP, 5'-AMP, and 3'-AMP. However, components of cAMP such as adenine and adenosine also are bound, indicating that the adenine moiety is the primary positive determinant of ligand binding. Specificity of cAMP binding appears to be established by hydrogen bonding interactions with the adenine base as well as by steric interactions with groups on the ribose moiety. In addition, the aptamer recognizes 8,5'-O-cycloadenosine (2) but not N(3), 5'-cycloadenosine (3), indicating that this RNA might selectively recognize the anti conformation of the N-glycosidic bond of cAMP.     

Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon

We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex.     

Bioassay of prostate-specific antigen (PSA) using microcantilevers

Diagnosis and monitoring of complex diseases such as cancer require quantitative detection of multiple proteins. Recent work has shown that when specific biomolecular binding occurs on one surface of a microcantilever beam, intermolecular nanomechanics bend the cantilever, which can be optically detected. Although this label-free technique readily lends itself to formation of microcantilever arrays, what has remained unclear is the technologically critical issue of whether it is sufficiently specific and sensitive to detect disease-related proteins at clinically relevant conditions and concentrations. As an example, we report here that microcantilevers of different geometries have been used to detect two forms of prostate-specific antigen (PSA) over a wide range of concentrations from 0.2 ng/ml to 60 microg/ml in a background of human serum albumin (HSA) and human plasminogen (HP) at 1 mg/ml, making this a clinically relevant diagnostic technique for prostate cancer. Because cantilever motion originates from the free-energy change induced by specific biomolecular binding, this technique may offer a common platform for high-throughput label-free analysis of protein-protein binding, DNA hybridization, and DNA-protein interactions, as well as drug discovery.     

The identification of novel RNA structural motifs using COMPADRES: an automated approach to structural discovery

Recurring RNA structural motifs are important sites of tertiary interaction and as such, are integral to RNA macromolecular structure. Although numerous RNA motifs have been classified and characterized, the identification of new motifs is of great interest. In this study, we discovered four new conformationally recurring motifs: the pi-turn, the Omega-turn, the alpha-loop and the C2'-endo mediated flipped adenosine motif. Not only do they have complex and interesting structures, but they participate in contacts of high biological significance. In a first for the RNA field, new motifs were discovered by a fully automated algorithm. This algorithm, COMPADRES, utilized a reduced representation of the RNA backbone and was highly successful at discerning unique structural relationships. This study also shows that recurring RNA substructures are not necessarily accompanied by consistent primary or secondary structure.     

The molecular pruning of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen

To identify the pharmacophore of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen (PSMA), a small analog library was designed and screened for inhibitory potency against PSMA. The design of the lead inhibitor was based upon N-acyl derivatives of endogenous substrate folyl-gamma-Glu and incorporates a phosphoramidate group to interact with the PSMA catalytic zinc atoms. The scope of the analog library was designed to test the importance of various functional groups to the inhibitory potency of the lead phosphoramidate. The IC(50) for the lead phosphoramidate inhibitor was 35 nM while the IC(50) values for the analog library presented a range from 0.86 nM to 4.1 microM. Computational docking, utilizing a recently solved X-ray crystal structure of the recombinant protein, along with enzyme inhibition data, was used to propose a pharmacophore model for the PSMA active site.