In vitro selection of ribozymes dependent on peptides for activity

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).     

Retention of conformational flexibility in HIV-1 Rev-RNA complexes

Sequence-specific recognition between HIV-1 Rev and viral RNA mediates the nuclear export of the viral mRNA and thus is important for the viral life cycle. HIV Rev binds to its viral RNA target with high affinity and specificity and also binds to an in vitro selected RNA aptamer that has a significantly different sequence from the viral RNA target with a 6-fold higher affinity than its natural target. The high-resolution structures of HIV Rev Arg-rich motif (ARM) in complexes with the wild-type RNA and the RNA aptamer reveal that, despite the significantly different RNA sequences, the two complexes share similar structural features and the protein-RNA interactions are mediated mostly by the Arg side chains in Rev ARM. To gain further insight into the role of these Arg side chains in the sequence-specific protein-RNA recognition, we have characterized the flexibility of these Arg side chains at the interfaces of the two high-affinity complexes using (15)N R(1), R(2), nuclear Overhauser effect, and chemical-shift anisotropy dipolar cross-correlation relaxation measurements. The ARM peptide contains uniformly (13)C/(15)N-labeled Arg residues, and the RNA samples were unlabeled. Despite the apparently similar roles of Arg side chains in both complexes, most of them display a different dynamic behavior in the context of different RNA molecules, and extensive and highly diverse motions have been observed for all of these side chains that interact with RNA. Most of the differences in side-chain dynamics between the complexes cannot be inferred from the three-dimensional structures. Additionally, more than half of the residues have increased flexibility in the Rev-RNA aptamer complex that has a higher affinity. This study provides new insights into ARM-RNA recognition and indicates that retention of conformational flexibility is likely important in high-affinity ARM-RNA recognition.     

Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.     

Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev

The Rev protein of HIV-1 regulates the synthesis of partially spliced forms of cytoplasmic viral mRNA by binding to a cis-acting RNA sequence, the Rev response element (RRE). We have investigated the regulation of splicing in vitro and have shown that Rev specifically inhibits splicing of pre-mRNAs containing an RRE by 3- to 4-fold. A synthetic peptide of 17 amino acids containing the RNA-binding domain of Rev is highly functional and specifically inhibits splicing by up to 30-fold. Other peptides that bind to the RRE with high affinity, but with low specificity, do not specifically inhibit splicing. Six repeated monomeric binding sites for the peptide can substitute for the RRE, indicating that regulation by Rev requires interactions with multiple sites. The peptide acts at a step in the assembly of splicing complexes, suggesting that one of the functions of the basic region of Rev is to prevent formation of a functional spliceosome.     

Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1′-Carbonyldiimidazole

A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1′-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.     

A primer-based approach to genome walking

A genome walking strategy based on annealing and ligation of single-stranded DNA primers to 3′ overhangs following restriction endonuclease digestion was developed. A set of primers contains 4 nucleotides at the 3′ end that are complementary to overhangs formed by restriction endonucleasesApaI;PstI;SacI andSphI. Following ligation, 5′ end overhangs are formed on the DNA, which serves as sites for the adaptor primers and nested primers for PCR amplification in combination with the gene-specific primers. This strategy was verified by the amplification of up to 4 kb of a potato leafroll virus full-length infectious clone. The procedure could be adopted to target any upstream and downstream regions flanking known sequences within the plant genome.     

Cleavage of RNA in hybrid duplexes by theE. coli ribonuclease H: II. Substrate properties of oligonucleotides containing nonnucleotide linkers

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for theE. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2–1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3′-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3′-3′-phosphodiester bond at the 3′-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity. For the previous report, see [1].     

Novel Strategies to Construct Complex Synthetic Vectors to Produce DNA Molecular Weight Standards

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies—sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and “small fragment accumulation”—for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field. Zhe Chen and Jianbing Wu contributed to this work equally.     

Allosteric aptamers: targeted reversibly attenuated probes

Aptamers are unique nucleic acids with regulatory potentials that differ markedly from those of proteins. A significant feature of aptamers not possessed by proteins is their ability to participate in at least two different types of three-dimensional structure: a single-stranded folded structure that makes multiple contacts with the aptamer target and a double-helical structure with a complementary nucleic acid sequence. We have made use of this structural flexibility to develop an aptamer-based biosensor (a targeted reversibly attenuated probe, TRAP) in which hybridization of a cis-complementary regulatory nucleic acid (attenuator) controls the ability of the aptamer to bind to its target molecule. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regNA) because it regulates the activity of the aptamer in the TRAP by hybridization with the central (intervening) sequence. The studies reported here of the ATP-DNA TRAP suggest that, as well as inhibiting the aptamer, the attenuator also acts as a structural guide, much like a chaperone, to promote proper folding of the TRAP such that it can be fully activated by the regDNA. We also show that activation of the aptamer in the TRAP by the complementary nucleic acid at physiological temperatures is sensitive to single-base mismatches. Aptamers that can be regulated by a specific nucleic sequence such as in an mRNA have potential for many in vivo applications including regulating a particular enzyme or signal transduction pathway or imaging gene expression in vivo.     

Aptameric enzyme subunit for homogeneous DNA sensing

We have developed an aptameric enzyme subunit (AES) which can detect the DNA in a homogeneous solution. The AES is an artificial enzyme subunit composed of an enzyme-inhibiting aptamer bearing a target-molecule binding site. We connected a probe DNA to a thrombin-inhibiting aptamer at its 5′ or 3′ end. The inhibitory activity of the thrombin-inhibiting aptamer bearing the probe DNA decreased compared to that of the original aptamer; however, it recovered upon hybridization with the target DNA. Using this AES, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution. K. Ikebukuro and W. Yoshida have contributed equally to this work.     

The role of reactivity in DNA templated native chemical PNA ligation during PCR

DNA templated fluorogenic reactions have been used as a diagnostic tool for the sequence specific detection of nucleic acids; and it has been shown that the native chemical ligation between thioester- and 1,2-aminothiol-modified PNA probes is amongst the most selective DNA detection methods reported. We explored whether a DNA templated reaction can be interfaced with the polymerase chain reaction (PCR). This endeavor posed a significant challenge. The reactive groups involved must be sufficiently stable to tolerate the high temperature applied in the PCR process. Nevertheless, the ligation reaction must proceed very rapidly and sequence specifically within the short time available in the annealing and primer extension steps before denaturation is used after approx. 1 min to commence the next PCR cycle. This required a careful optimization of the ternary complex architecture as well as adjustments of probe length and probe reactivities. Our results point to the prime importance of the ligation architecture. We show that once suitable annealing sites have been identified less reactive probe sets may be preferable if sequence specificity is of major concern. The reactivity tuning enabled the development of an in-PCR ligation, which was used for the single nucleotide specific typing of the V600E (T1799A) point mutation in the human BRaf gene. Showcasing the efficiency and sequence specificity of native chemical PNA ligation, attomolar template proofed sufficient to trigger signal while a 1000-fold higher load of single mismatched template failed to induce appreciable signal.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

Development of RNA aptamer that inhibits methyltransferase activity of dengue virus

Objectives

To develop an RNA aptamer specific for the methyltransferase (MTase) of dengue virus (DENV) which is essential for viral genome replication and translation acting directly on N-7 and 2′-O-methylation of the type-I cap structure of the viral RNA.

Results

We identified 2′-fluoro-modified RNA aptamers that can specifically bind DENV serotype 2 (DENV2) MTase using systematic evolution of ligands by exponential enrichment technology. We truncated the chosen aptamer into a 45-mer RNA sequence that can bind DENV2 MTase with K _d  ~ 28 nM and inhibit N-7 methylation activity of the protein. Moreover, the 45-mer truncated aptamer could not only bind with an K _d  ~ 15.6 nM but also inhibit methylation activity of DENV serotype 3 (DENV3) MTase. The 45-mer aptamer competitively impeded binding of both DENV2 and DENV3 genomic RNA to MTase of each serotype.

Conclusion

The selected 45-mer truncated RNA aptamer specifically and avidly bound DENV MTase and competitively inhibited its methylation activity, and thus could be useful for the development of anti-DENV agents.

     

Construction of HIV Rev peptides containing peptide nucleic acid that bind HIV RRE IIB RNA

Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.     

Thermodynamic and Kinetic Characterization of Duplex Formation between 2′-O, 4′-C-Methylene-modified Oligoribonucleotides,DNA and RNA

2′-O,4′-C-methylene-linked ribonucleotide derivatives, named LNA (locked nucleic acid) and BNA (bridged nucleic acid) are nucleic acid analogoues that have shown high-affinity recognition of DNA and RNA, and the employment of LNA oligomers for antisense activity, gene regulation and nucleic acid diagnostics seems promising. Here we show kinetic and thermodynamic results on the interaction of a series of 10 bases long LNA–DNA mixmers, gabmers as well as full length LNA’s with the complementary DNA, RNA and LNA oligonucleotides in the presence and absence of 10 mM Mg²⁺- ions. Our results show no significant differences in the reaction thermodynamics and kinetics between the LNA species, only a tendency to stronger duplex formation with the gabmer and mixmer. Introduction of a few LNA’s thus may be a better strategy, than using full length LNA’s to obtain an oligonucleotide that markedly increases the strength of duplexes formed with the complementary DNA and RNA.     

Solution Structure of a Novel C<Subscript>2</Subscript>-Symmetrical Bifunctional Bicyclic Inhibitor Based on SFTI-1

A novel bifunctional bicyclic inhibitor has been created that combines features both from the Bowman–Birk inhibitor (BBI) proteins, which have two distinct inhibitory sites, and from sunflower trypsin inhibitor-1 (SFTI-1), which has a compact bicyclic structure. The inhibitor was designed by fusing together a pair of reactive loops based on a sequence derived from SFTI-1 to create a backbone-cyclized disulfide-bridged 16-mer peptide. This peptide has two symmetrically spaced trypsin binding sites. Its synthesis and biological activity have been reported in a previous communication [Jaulent and Leatherbarrow, 2004, PEDS 17, 681]. In the present study we have examined the three-dimensional structure of the molecule. We find that the new inhibitor, which has a symmetrical 8-mer half-cystine CTKSIPP′I′ motif repeated through a C₂ symmetry axis also shows a complete symmetry in its three-dimensional structure. Each of the two loops adopts the expected canonical conformation common to all BBIs as well as SFTI-1. We also find that the inhibitor displays a strong and unique structural identity, with a notable lack of minor conformational isomers that characterise most reactive site loop mimics examined to date as well as SFTI-1. This suggests that the presence of the additional cyclic loop acts to restrict conformational mobility and that the deliberate introduction of cyclic symmetry may offer a general route to locking the conformation of β-hairpin structures. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Analysis of RNA Silencing in Agroinfiltrated Leaves of <Emphasis Type="Italic">Nicotiana Benthamiana</Emphasis> and <Emphasis Type="Italic">Nicotiana Tabacum</Emphasis>

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.     

Development of a novel pretargeting system with bifunctional nucleic acid molecules

This study was aimed at exploring a novel pretargeting system based upon bifunctional nucleic acid molecules that are comprised of a nucleic acid aptamer and a nucleic acid tail. The properties of bifunctional molecules were investigated by both theoretical prediction and experimental determination. Different from the algorithm-based structure prediction, the experimental data showed that some nucleic acid tails could significantly decrease the binding capability of the aptamer. It was also found that the effectiveness of bifunctional molecules in labeling cells was dependent on the hybridization length. Based on these understandings, one bifunctional molecule was selected to study pretargeting. The results demonstrated that the bifunctional molecule could not only bind to target cells, but also hybridize with its complementary oligonucleotide on the cell surface. Thus, bifunctional nucleic acid molecules hold great potential for pretargeting applications.