Biomedical subjects

Donald H Burke

Publications and source records attributed to Donald H Burke.

At least 19 recordsLinked to original sources

PubMed2006

Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme.

The Kin.46 ribozyme catalyzes transfer of the gamma (thio)phosphoryl group of ATP (or ATPgammaS) to the ribozyme's 5' hydroxyl. Single-turnover catalytic activities of topologically rearranged versions of Kin.46 were studied to gain insight into its overall tertiary architecture. The distal ends of stems P3 and P4 were tethered through a single-stranded connection domain that altered the interhelical connectivity. The shortest linkers interfered with catalysis, while seven or more nucleotides (nt) in the linker allowed near-normal catalytic rates, suggesting that a distance of roughly 25-35 A optimally separates the termini of these helices. Activity was maximal when the tether contained 15 nt, at which point the k(cat) (0.016 min(-1)) and Km (1.2 mM) values were identical to those of a nontethered control. The presence of the tether alters Mg(2+) dependence, in that Mg2+ binding appears to be more cooperative in the tethered ribozyme (Hill coefficient 1.4-1.8 versus 0.8 for the nontethered ribozyme). Binding affinity for the ATPgammaS substrate increases at elevated concentrations of Mg2+, particularly for the tethered ribozyme. The tethered ribozyme displays significantly enhanced thermal stability, with a maximum initial velocity (0.126 min(-1)) at 60 degrees C, whereas the nontethered ribozyme has a lower maximum initial velocity (0.051 min(-1)) at 50 degrees C. The tether also significantly reduces the apparent entropy of activation. Both of these effects can be understood in terms of stabilization of the ribozyme in a conformation that is on-path with respect to catalysis, and in terms of facilitating formation of the allosteric activation helix P4.

Adenosine Triphosphate↗

PubMed2006

Extraordinary rates of transition metal ion-mediated ribozyme catalysis.

In pre-steady-state, fast-quench kinetic analysis, the tertiary-stabilized hammerhead ribozyme "RzB" cleaves its substrate RNA with maximal measured k (obs) values of approximately 3000 min(-1) in 1 mM Mn(2+) and approximately 780 min(-1) in 1 mM Mg(2+) at 37 degrees C (pH 7.4). Apparent pKa for the catalytic general base is approximately 7.8-8.5, independent of the corresponding metal hydrate pKa, suggesting potential involvement of a nucleobase as general base as suggested previously from nucleobase substitution studies. The pH-rate profile is bell-shaped for Cd(2+), for which the general catalytic acid has a pKa of 7.3 +/- 0.1. Simulations of the pH-rate relation suggest a pKa for the general catalytic acid to be approximately 9.5 in Mn(2+) and >9.5 in Mg(2+). The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by approximately 1-2 pH units in the presence of Cd(2+) and Mn(2+). One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid.

Base Sequence↗

PubMed2006

Differential susceptibility of HIV-1 reverse transcriptase to inhibition by RNA aptamers in enzymatic reactions monitoring specific steps during genome replication.

Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC50 values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA-dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.

Cloning, Molecular↗

PubMed2006

Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.

Ribozymes that phosphorylate internal 2'-OH positions mimic the first mechanistic step of P-type ATPase enzymes by forming a phospho-enzyme intermediate. We previously described 2'-autophosphorylation and autothiophosphorylation by the 2PTmin3.2 ribozyme. In the present work we demonstrate that the thiophosphorylated form of this ribozyme can de-thiophosphorylate in the absence of ATPgammaS. Identical ionic conditions yield a thiophosphorylated strand when ATPgammaS is included, thus effecting a net ATPgammaS hydrolysis. The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions. By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1). The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

Adenosine Triphosphatases↗

PubMed2006

HIV-1 inactivation by nucleic acid aptamers.

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.

Acquired Immunodeficiency Syndrome↗

PubMed2005

A trans acting ribozyme that phosphorylates exogenous RNA.

The structural complexity required for substrate recognition within an active site constrains the evolution of novel catalytic functions. To evaluate those constraints within populations of incipient ribozymes, we performed a selection for kinase ribozymes under conditions that allowed competition for phosphorylation at nine candidate sites. Two candidate sites are the hydroxyl groups on a "quasi-diffusible" chloramphenicol (Cam) moiety tethered to the evolving library through an inert, flexible linker. A subtractive step was included to allow only seven ribose 2' hydroxyls to compete with the two Cam hydroxyls for phosphorylation. After the library was incubated with gamma-thio-ATP (ATPgammaS), active species were recovered from a polyacrylamide gel containing [(N-acryloylamino)phenyl] mercury (APM) and amplified for further cycles of selection. Activity assays on selected isolates and truncated derivatives identified the essential secondary structure of the dominant RNA motif. Phosphorylation was independent of the Cam moiety, indicating ribose 2' phosphorylation. The dominant motif was separated into catalytic "ribozyme" and "substrate" strands. Partial alkaline digestion of the substrate strand before and after phosphorylation identified the precise modification site as the first purine (R) within the required sequence 5'-RAAAANCG-3'. The reaction shows approximately 10-fold preference for ATPgammaS over ATP and is independent of pH over a wide range (5.5-8.9), consistent with a dissociative reaction mechanism that is rate-limited by formation of a metaphosphate transition state. Divalent metal ions are required, with a slight preference of Mn(2+) > Mg(2+) > Ca(2+). Lack of reactivity in [Co(NH(3))(6)](3+) indicates a requirement for inner sphere contact with the metal ion, either for structural stabilization, catalysis, or both.

Adenosine Triphosphate↗

PubMed2005

Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities.

BACKGROUND: Low concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization. RESULTS: Stem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl2 concentrations as low as 0.2 mM at 25 degrees C and 0.5 mM at 37 degrees C. Under multiple-turnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl2 concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active. CONCLUSION: Hammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the stabilization and substrate recognition functions of stem 1 increases cleavage activity at physiological concentrations of divalent magnesium while retaining recognition of exogenous targets. Trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of all natural hammerhead topologies can therefore be used in intracellular applications.

Enzyme Activation↗

PubMed2004

Deprotonation stimulates productive folding in allosteric TRAP hammerhead ribozymes.

Hammerhead ribozymes in crystals change conformation in response to deprotonation of the nucleophilic 2' OH, thereby aligning the hydroxyl for in-line displacement at the scissile phosphate. Published data do not address whether deprotonation affects folding in solution. Allosteric hammerhead "TRAPs," when activated by the appropriate oligonucleotide, show the expected log-linear relation between initial cleavage rate and pH. In contrast, attenuated TRAPs shows biphasic kinetics in which a rapid burst is followed by slow cleavage that is nearly independent of pH. Attenuated ribozymes are stimulated by urea at both low and high pH, confirming that rearrangement of secondary structure is rate-limiting for the attenuated ribozymes once they have folded. Plots of burst magnitude versus pH in the absence of urea show a sharp transition around pH 8.3, which is near the kinetic pKa for the cleavage reaction in Mg2+. Raising the pH after folding at pH 7.5 did not activate attenuated ribozymes even when the RNA was incubated at the elevated pH for extended periods prior to addition of Mg2+. In contrast, lowering the pH after folding at pH 9.5 rapidly re-established attenuation. Deprotonation of the ribozyme-substrate complex thus appears to alter the folding landscape such that a metastable "pre-activated" complex forms before the thermodynamically more stable attenuated state can be attained. From the initial partition into active and inactive conformers, we estimate that this deprotonation contributes approximately 1.2 kcal/mol toward stabilization of the active fold at a crucial step during folding of the TRAP. Assuming that the nucleophilic 2' OH is the relevant acid, its deprotonation would thus serve a dual role of favoring productive fold and enhancing the nucleophilicity of this oxygen.

Allosteric Site↗

PubMed2004

Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry.

We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [(N-acryloylamino)phenyl] mercuric chloride (APM) polyacrylamide gels, whereas millimolar concentrations of TCEP gave no difference in the fraction of thiophosphorylated RNA retained on the APM interface relative to samples containing no reductant. Ribozyme activity in TCEP, assessed by the self-thiophosphorylating Kin.46 ribozyme, was unaffected by the presence of DTT or TCEP or by the absence of reductant, as measured on APM gels and evaluated by Michaelis-Menten kinetics. Unexpectedly, TCEP more than doubled the half-life of full-length RNA at 50 and 70 degrees C, whether in 5 or 50mM MgCl(2), relative to DTT and the absence of reductant. Under these same conditions, the 5(')-thiophosphate showed negligible decay, and TCEP was more stable than DTT. TCEP thermostability was equivalent in the presence of 5 or 50mM MgCl(2) and 10mM adenosine or ATP.

Base Sequence↗

PubMed2004

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures.

Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 microM MgCl2. The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80 degrees C in 10 mM MgCl2 and at 70 degrees C in 2 mM MgCl2. A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl2, including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.

Base Sequence↗

PubMed2003

The tyranny of adenosine recognition among RNA aptamers to coenzyme A.

BACKGROUND: Understanding the diversity of interactions between RNA aptamers and nucleotide cofactors promises both to facilitate the design of new RNA enzymes that utilize these cofactors and to constrain models of RNA World evolution. In previous work, we isolated six pools of high affinity RNA aptamers to coenzyme A (CoA), the principle cofactor in biological acyltransfer reactions. Interpretation of the evolutionary significance of those results was made difficult by the fact that the affinity resin attachment strongly influenced the outcome of those selections. Here we describe the selection of four new pools isolated on a disulfide-linked CoA affinity matrix to minimize context-dependent recognition imposed by the attachment to the solid support. RESULTS: The four new aptamer libraries show no sequence or structural relation to a previously dominant CoA-binding species, even though they were isolated from the same initial random libraries. Recognition appears to be limited to the adenosine portion of the CoA--in particular the Höogsteen edge--for most isolates surveyed, even when a counter selection was employed to remove such RNAs. Two of the recovered isolates are eluted with intact CoA more efficiently than with AMP alone suggesting a possible pantotheine interaction. However, a detailed examination of recognition specificity revealed that the 3' phosphate of CoA, and not the pantotheine arm, determined recognition by these two isolates. CONCLUSION: Most aptamers that have been targeted towards cofactors containing adenosine recognize only the adenosine portion of the cofactor. They do not distinguish substituents on the 5' carbon, even when those substituents have offered hydrogen bonding opportunities and the selection conditions discouraged adenosine recognition. Beyond hydrogen bonding, additional factors that guide the selection towards adenosine recognition include aromatic stacking interactions and relatively few rotational degrees of freedom. In the present work, a sterically accessible, disulfide-linked CoA affinity resin afforded the selection of a more diverse aptamer collection then previous work with a N6 linked CoA resin.

Adenosine↗

PubMed2003

Structural flexibility and the thermodynamics of helix exchange constrain attenuation and allosteric activation of hammerhead ribozyme TRAPs.

Perturbations of precleavage equilibria in RNA-cleaving ribozymes can be exploited to control cleavage kinetics. In the targeted ribozyme-attenuated probes (TRAP) design, antisense and attenuator sequences are appended onto the catalytic core of a ribozyme or deoxyribozyme. The attenuator anneals to conserved bases in the catalytic core to form an inactive conformation, which is activated upon binding of a sense strand oligonucleotide to the antisense module. In this work, the apparent Michaelis-Menton constant (K'm) for the binding of the RNA substrate to the ribozyme is shown to be within a factor of 2 for a number of constructs whose observed cleavage rates varied by several 100-fold. These observations rule out models of allosteric regulation based on modulation of substrate binding affinity, instead favoring a model in which regulation arises from equilibration between the active and inactive conformations of the TRAP. Free energies of formation for isolated helices that are exchanged during this reequilibration were determined from the concentration dependence of optical melt data. These values established that the thermodynamic stabilities of sense-antisense duplexes and of the attenuator-core duplexes correlate with observed rates of cleavage. Notably reduced cleavage rates are observed for TRAP ribozymes with extended antisense sequences, suggesting that tight binding of attenuator to the core is assisted by a long antisense portion. A construct with a 25-nucleotide antisense showed greater than 730-fold activation upon annealing with a 20-nucleotide DNA sense strand oligo, representing the greatest activation observed to date for the TRAP design.

Allosteric Regulation↗

PubMed2003

Evolutionary landscapes for the acquisition of new ligand recognition by RNA aptamers.

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro--unconstrained by confounding biological factors--is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences--defined as the set's functional tolerance for point mutations--and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.

Base Sequence↗

PubMed2003

Nickel2+-mediated assembly of an RNA-amino acid complex.

The RNA species SHR1 reacts with biocytin (epsilon-biotinoyl-L-lysine) in the presence of Ni(2+) or Pt(2+) to produce a metal-bridged complex that migrates more slowly than unreacted RNA in the presence of streptavidin (StrAv) on denaturing polyacrylamide gels. Mapping of reverse transcription pause sites identified G79 as a reactive nucleotide. G79 is near the 3' end of a 37 nucleotide core motif that is nearly as reactive as SHR1. SHR1 reacts with biocytin in the presence of Pt(2+) to yield a product that comigrates with the Ni(2+) product but that is much more stable, suggesting that the metal ion used in the reaction is present in the product, possibly linking the RNA to the amino acid. In support of this model, SHR1 shows a strong affinity for Ni(2+) in immobilized metal ion chromatography.

Amino Acids↗

PubMed2003

Inhibition of HIV-1 reverse transcriptase by RNA aptamers in Escherichia coli.

A better understanding of aptamer function in bacteria would help to establish simple model systems for screening RNA-protein interactions within an intracellular context. Escherichia coli DNA polymerase I mutants (Pol I(ts)) fail to grow at 37 degrees C unless an exogenous DNA polymerase such as HIV-1 reverse transcriptase (RT) is expressed within the cell. Here, we show that four RNA aptamers that inhibit HIV-1 RT in vitro block complementation by HIV-1 RT when expressed in vivo. No other essential functions are impaired by aptamer expression at either temperature. Intracellular aptamer RNA concentrations from induced cultures were measured to range from 76 to 180 nM, which is comparable with exogenously expressed HIV-1 RT levels in these cells. RT polymerase activity was reduced to background levels in cell-free extracts prepared from cultures expressing both HIV-1 RT and the 70.28 aptamer, compared with extracts from cultures expressing HIV-1 RT alone. Intracellularly expressed RNA aptamers can thus be used to generate conditional null mutants in bacteria by titrating an essential protein.

Escherichia coli↗

PubMed2002

Allosteric hammerhead ribozyme TRAPs.

A new mode of allosteric regulation of nucleic acid enzymes is described and shown to operate effectively with hammerhead ribozymes. In the "TRAP" design (for targeted ribozyme-attenuated probe), a 3' terminal "attenuator" anneals to conserved bases in the catalytic core to form the "off" state of the ribozyme. Binding of RNA or DNA to an antisense sequence linking the ribozyme and attenuator frees the core to fold into an active conformation, even though the antisense sequence itself does not interfere with the ribozyme. TRAP hammerheads based on the previously characterized HH8 ribozyme were shown to be activated more than 250-fold upon addition of the sense strand. RNA oligonucleotides were more effective activators than DNA oligos, consistent with the known relative helix stabilities (RNA-RNA > RNA-DNA). Oligonucleotides that directly paired with the attenuator gave up to 1760-fold activation. The magnitude of the activation was greater when the oligo was added prior to folding than if it was added during the cleavage reaction. The TRAP design requires no prior knowledge of (deoxy)ribozyme structure beyond identification of the essential core. Thus, this approach should be readily generalizable to other systems for biomedicine, sensor technology, and additional applications.

Allosteric Regulation↗

PubMed2002

Boron-containing aptamers to ATP.

Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5'-(alpha-P-borano) triphosphate (bG) for GTP or uridine 5'-(alpha-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the zeta-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some zeta-fold aptamers, while the bG selection yielded none of this type. Non-zeta aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.

Adenosine Triphosphate↗

PubMed2002

Flavin recognition by an RNA aptamer targeted toward FAD.

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA-cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH(2), nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5-8.9). Since general acid-base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes.

Base Sequence↗