Prions met Aptamers
Mad Cow Home -- Best Links -- Search this site

RNA aptamers specifically interact with the prion protein
How aptamers might be used in diagnosis and therapy
Could aptamers be responsible for prion insertion mutations?
What is an apatamer anyway?
Show me a graphic of a stackable G-quartet

RNA aptamers specifically interact with the prion protein PrP.

J Virol 1997 Nov;71(11):8790-8797 
Weiss S, Proske D, Neumann M, Groschup MH, Kretzschmar HA, Famulok M, Winnacker EL
We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein 23-231 fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST:: 90-231, which lacks 67 amino acids from the PrP N terminus.

The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition.

Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice, hamsters, and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice. Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize rogue prion in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.

How aptamers might be used in diagnosis and therapy

[opinion --webmaster 31 Oct 97]
The authors note that their prion aptamer can specifically recognize the post-signal, pre-repeat region of normal prion protein in crude brain homogenates. This region is missing from rogue prion, which generally begins after residue 90. The number of oligonucleotide sequence variants screened can approach ten to the twentieth, with strong selection at each step. It would seem that a second aptamer could be developed that would be specific for this later stretch or specifically for rogue prion and its nucleating ends. This process could be hugely important in detecting contaminated blood: pooled blood products are run through a gel with covalently affixed aptamer. An aptamer could also be expressed fairly easily in situ, which has exciting prospects for therapy, as a non-toxic capping agent.

The binding pocket of an aptamers is built on (forget Watson-Crick and Donohue) Hoogsteen hydrogen bonds and a stackable element called a G-tetrad (or G-quartet) that comes up as well in telomeres and, I should say, as the pausing mechanism of DNA polymerase within the prion repeat region, giving rise to deletions and insertion mutations due to the repeat slippage mechanism. Tandem repeats involving polyglycine GGx are the proverbial accident waiting to happen.

There would be a certain irony here if the reagent used to eliminate prion agent was based on the same structure that caused so much of it. (Two percent of the population carries a deletion mutant -- a large new patient series by Cervenakova et al. suggests these may not be neutral after all.)

Backgrounder: snippets from various articles concerning aptamers

"...Starting from a RNA pool that had a random sequence core of 12-18 nucleotides, aptamers that bind specifically to the protein were selected after 10 rounds of selection and amplification. A single aptamer was found predominantly (71%) in the selected pool. This aptamer could bind to the protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro.

... RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 1014 different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.

... We subjected a pool of 1015 RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.

... In vitro selection experiments have produced nucleic acid ligands (aptamers) that bind tightly and specifically to a great variety of target biomolecules. The utility of aptamers is often limited by their vulnerability to nucleases present in biological materials. One way to circumvent this problem is to select an aptamer that binds the enantiomer of the target, then synthesize the enantiomer of the aptamer as a nuclease-insensitive ligand of the normal target.

... We have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control.

... RNA molecules were selected from a random sequence library for their ability to bind to an RNA stem-loop target. Oligonucleotides with extensive Watson-Crick complementarity to the RNA ligand were selected against by inclusion of a blocking oligodeoxynucleotide in the binding phase of the selection protocol. After 18 generations of SELEX (systematic evolution of ligands by exponential enrichment) a single RNA family was predominant in the binding population. The winning aptamer RNA bound the target RNA with an apparent Kd = 70 nM. Structural mapping and Fe(II)-EDTA protection indicated that the target RNA interacted with small unpaired loops in the aptamer structure.

... We used in vitro selection (SELEX) to isolate RNA 'aptamers' to S-adenosyl methionine (SAM). Individual aptamer sequences conform to the structural element noted previously for adenosine binding in selections for aptamers to ATP and NAD+. When we compare the patterns of sequence conservation among 65 adenosine-binding sequences to the published structure of the adenosine aptamer, we find that the most highly conserved nucleotides contact the bound adenosine directly, and that one conserved nucleotide outside the binding pocket is in position to stabilize nucleotides within the binding pocket. The aptamer's ability to bind diverse adenosine-containing cofactors is easily understood in terms of its mode of binding, which leaves the 5'position exposed to solvent. We propose that aptamers that bind their targets away from the reactive moiety may be particularly well suited for catalysis. Finally, we estimate that one sequence in 10(11) may be able to form this structural motif, and that there may be many other adenosine-binding motifs that have escaped detection because of their lower representation in the starting random pools.

... We have previously shown that the G-rich sequence G16CG(GGT)2GG in the promoter region of the chicken beta-globin gene poses a formidable barrier to DNA synthesis in vitro (Woodford et al., 1994, J. Biol. Chem. 269, 27029-27035). The K+ requirement, template-strand specificity, template concentration independence, and involvement of Hoogsteen bonding suggested that the underlying basis of this new type of DNA synthesis arrest site might be an intrastrand tetrahelical structure. However, the arrest site lacks the four G-rich repeats that are a hallmark of previously described intramolecular tetraplexes and contains a number of noncanonical bases that would be expected to greatly destabilize such a structure. Here we report evidence for an unusual K+-dependent intrastrand "cinched" tetraplex.

... A DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming Hoogsteen pairs with purines of the Watson-Crick basepairs. The same modeling method was used to investigate the feasibility of three-dimensional structures based on the three possible alternative hydrogen-bonding schemes: Watson-Crick-reverse Hoogsteen, Donohue [J. Donohue (1953) Proceedings of the National Academy of Science USA, Vol. 39, pp. 470-475] (reverse Watson-Crick)-Hoogsteen, and Donohue-reverse Hoogsteen. We found that none of these can occur in either RNA or DNA helices because they give rise only to structures with prohibitively short contacts between backbone and base atoms in the same chain.

... There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment. G2T4CG2 with Na cation as counterion forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation

... The thrombin aptamer is a single-stranded DNA of 15 nucleotides that was identified by the selection of thrombin-binding molecules from a large combinatorial library of oligonucleotides. This prototype aptamer of thrombin has a unique double G-tetrad structure capable of inhibiting thrombin at nanomolar concentrations. Substitution of arginine 70 in thrombin exosite I with glutamic acid effectively eliminated binding of the prototype thrombin aptamer.

... There is increasing evidence that four-stranded Hoogsteen-bonded DNA structures, G4-DNA, play an important role in cellular processes such as meiosis and recombination. The Hoogsteen-bonded G4-DNA is thermodynamically more stable than duplex DNA, and many guanine-rich genomic DNA sequences with the ability to form G4-DNA have been identified. A protein-dependent activity that resolves G4-DNA into single-stranded DNA has been identified in human placental tissue. The products of the resolvase reaction are unmodified single-stranded DNA. The resolvase is not a duplex DNA helicase or a topoisomerase II activity and does not unwind Hoogsteen-bonded triplex DNA. Resolvase is a novel activity that unwinds stable G4-DNA structures using a dNTP-dependent mechanism producing unmodified single-stranded DNA.

... A G-tetrad can exhibit considerable stability, comparable to or even exceeding that of most Watson-Crick nearest-neighbor interactions, with this stability resulting from a very favorable enthalpy of formation. The observed NOEs between the amino and H8 protons on adjacent guanines within individual G-tetrads support the Hoogsteen pairing alignment around the tetrad.

... Telomeres are structures at the ends of eukaryotic chromosomes, the DNA of which contains stretches of tandemly repeated sequences with G clusters along one strand. Model telomeric G-rich DNAs can form different tetraplex structures, stabilized by cyclic hydrogen bonding of four guanines in the presence of metal ions such as Na+ or K+. Oligonucleotides with a single copy of the Oxytricha sequence dT4G4 form a tetramer, with a parallel-stranded, right-handed helical structure. Additional copies favor folded-back structures that associate to form an antiparallel dimer. The parallel-stranded tetramer has all G's in the anti configuration, while the folded-back dimer has alternating syn and anti nucleotide conformations along each strand.

... Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.

... Guanine mono-, oligo-, and polynucleotides, including the guanine-rich telomeric sequences found at the ends of chromosomes, have been shown to form self-associated species which contain cyclic tetramers of hydrogen-bonded guanines (G-tetrads).

... The telomeric DNA of Tetrahymena consists of T2G4 repeats, and models have been previously proposed for the intramolecular folded structure of the d(T2G4)4 sequence based on chemical footprinting and cross-linking data. A high-resolution solution structure of this sequence would allow comparison with the structures of related G-tetraplexes. T2G4)4 folds intramolecularly into a right-handed G-tetraplex containing three stacked G-tetrads connected by linker segments consisting of a G-T-T-G lateral loop, a central T-T-G lateral loop and a T-T segment that spans the groove through a double chain reversal. The latter introduces a new G-tetraplex folding topology with unprecedented combinations of strand directionalities and groove widths, as well as guanine syn/anti distributions along individual strands and around individual G-tetrads. The observed structural polymorphism establishes that G-tetraplexes can adopt topologies which project distinctly different groove dimensions, G-tetrad base edges and linker segments for recognition by, and interactions with, other nucleic acids and proteins.

... A 100-fold higher affinity for this winner sequence sequence versus either a randomized version of this sequence or a 20-mer oligo corresponding to an unrelated beta-globin intron sequence. This winner RNA oligo aggregates in solution to form an apparent dimer that may represent a G-quartet resulting from dimerization of two Hoogsteen base-paired hairpins.

... The sequences G3T2A 3G3 and T2AG34 assume an antiparallel G quartet structure by intramolecular folding, while the sequence G3T2AG3 also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide G3T2AG3 forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes.

... A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin.

... This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T.T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.

... This DNA adopts a highly compact, highly symmetrical structure which consists of two tetrads of guanosine base pairs and three loops. The residues of the tetrads alternate anti-syn-anti-syn. This novel structural motif for DNA may also be relevant to the structure of telomere DNA.

... Aptamers are double-stranded DNA or single-stranded RNA molecules that bind specific molecular targets. Large randomly generated populations can be enriched in aptamers by in vitro selection and polymerase chain reaction. But so far single-stranded DNA has not been investigated for aptamer properties, nor has a target protein been considered that does not interact physiologically with nucleic acid. Here we describe the isolation of single-stranded DNA aptamers to the protease thrombin of the blood coagulation cascade and report binding affinities in the range 25-200 nM. Sequence data from 32 thrombin aptamers, selected from a pool of DNA containing 60 nucleotides of random sequence, displayed a highly conserved 14-17-base region. Several of these aptamers at nanomolar concentrations inhibited thrombin-catalysed fibrin-clot formation in vitro using either purified fibrinogen or human plasma."

Example of two G-tetrads (thrombin aptamer: get full pdb of 148D ) or just partial

Mad Cow Home or Best Links