The regulation of transcription elongation is important in both prokaryotic and eukaryotic gene regulation (1-5). However, much less is known about the regulation of elongation than of initiation, even in classical systems such as bacteriophage , in which termination (6) and elongation regulation were first described (7, 8). The N protein is a key regulator of transcription elongation in phage (9-11), interacting with (i) the nascent mRNA (ii) the transcription factor NusA and (iii) RNA polymerase, despite being only 107 amino acids long and totally unfolded in solution (Figure 5.1a,b) (12). N increases the processivity of RNA polymerase, allowing it to read through both intrinsic and Rho-dependent termination signals (9-11), and initiates this antitermination activity by specifically binding the nascent mRNA at the boxB stem loop within the sequence known as nut (N-utilization) (13, 14) (Figure 5.1b,c). Despite recent crystallographic work describing prokaryotic transcription machinery (15), various features of this antitermination switch remain mysterious. For example, controversy exists over whether approximation of components in the antitermination machinery is sufficient for function (16) or whether the detailed conformations of cis-acting elements (17) have any role.
Phage display involves the insertion of a gene encoding a pro- tein of interest into a phage coat protein gene, causing the phage to display the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype (Fig. 2f). In this way, by immobilizing an RNA target to beads or to the surface of a microtiter plate well, large protein libraries can be screened against the RNA target and ampliﬁed in a process called in vitroselection. Phage display has been successfully used to investigate the RNA-binding speciﬁcity of the mammalian spliceosomal protein U1A, which binds to hair- pin II of the U1 small nuclear RNA (U1hpII) , as well as to iso- late single zinc ﬁngers that bind complex RNA structures with high afﬁnity and speciﬁcity . A number of phage libraries display- ing cyclic peptides and linear peptides are commercially available. However, the size of the libraries is limited by the transformation efﬁciency of bacterial cells and only a limited number of individual clones can be examined easily making this method time-consum- ing, difﬁcult to scale-up and labour-intensive. mRNA display, also called mRNA–protein fusion  or in vitro virus , relies on the covalent coupling of mRNA to the nascent polypeptide (Fig. 2g). Numerous mRNA-display selections have isolated more than one hundred chemically distinct RNA-bindingpeptides . Most of the experiments have used the RNA-binding domain from phage k N protein owing to its small size and high afﬁnity for its cognate RNA. Despite its advantages, mRNA display has some limitations. The major concerns are the possibilities that the cova- lently attached mRNA interferes with the function of the protein, or that the target RNA interacts with the displayed protein. Other weaknesses of mRNA display are limitations in the display of mem- brane-bound proteins owing to their low expression level in in vitro translation systems  and of proteins whose biological functions relies on complex formation.
Gene expression constructs encoding recombinant L. laeta SMDs Ll1 and Ll2 were generated. The 858 bp cDNAs for the mature form of SMD-Ll1 [GenBank:DQ369999] and SMD-Ll2 [GenBank:DQ37000]  were assembled in vitro by polymerase chain assembly, which employs long synthetic oligonucleotides as starting material for extension and amplification reactions ; Pfu DNA polymerase (Fermentas) was used. For each SMD, 22 overlapping oligonucleotides were designed (overlaps of 18 nts): 2 external oligonucleotides (33 nts each) and 20 internal oligonucleotides (59 nts each), all synthesized by Integrated DNA Technologies (IDT). Cloning schemes demanded introducing single nucleotide modifications as well as the addition of AAG CTT ACT G to the 3′ end to incorporate a HindIII site. Specifically, for SMD-Ll1, a nonsilent mutation 26A > T (N9I), and four silent muta- tions (36 T > C, 462 T > C, 618C > T, 837A > T); for SMD- Ll2, five silent mutations (141 T > C, 300 T > C, 462 T > C, 618C > T, 837A > T). Each cDNA was assembled in two halves from which full-length sequences (868 bp) were ob- tained. Amplicons were purified from agarose gels with the Wizard SV Gel and PCR Clean-up System (Promega). The sequences required for protein production and purifi- cation were provided by directional cloning (blunt end/ HindIII) in pQE30-Xa (Qiagen) (StuI, Klenow, HindIII). The EcoRI-HindIII fragments from these intermediate constructs contained sequences encoding a ribosome binding site, an initiation codon, and a recombinant pro- tein having a His 6 tag, the four residues required for Figure 4 Cross effects of leader Gs12 aptamers on SMD-Ll1
The in vitroselection cycle shown in Figure 2.1 will be applied to the dienes and dienophiles synthesized in this project. The selection process will use RNA containing 5-(carboxamide-4-pyridylmethyl)-uridine triphosphate and selected main group metal ions (Na + , K + , Mg 2+ , and Ca 2+ ) and transition metal ions (Cu(II), Mn(II), Co(II), Ni(II), Fe(II), and Zn(II)). The pyridine modification in combination with the metal ions has been shown to create a unique metal binding site capable of Lewis acid catalysis in other systems. 6 In the first round of selection, a library of > 10 14 random RNA sequences that have been covalently linked to a diene substrate through a polyethylene glycol (PEG) linker containing a cleavage (CL) site, will be incubated with an aldehyde dienophile covalently bound to a biotin anchor group. The RNAs that successfully catalyze the HDA reaction will be bound to the biotin anchor group. These RNA species will be separated from the unreacted RNA species by a streptavidin partitioning step.
otides 300 and 320 is at least one important binding partner of L22 in vivo. First, independent isolates of RNA fragments comprising this region were obtained most often, and the rel- ative levels of affinity of these RNA fragments appear to be higher than those of other RNA sequences isolated by cDNA- SELEX (Fig. 6). Second, this region is highly conserved among various eukaryotic species and includes the three residues ap- parently required for tight binding to L22, and its folding into a stem-loop structure in vitro and in vivo has been previously predicted (2, 34). The sequences of ribosomal RNA within this region are 100% identical for humans, Mus musculus, Rattus norvegicus, Xenopus laevis, and Drosophila melanogaster; the Saccharomyces cerevisiae large subunit RNA has a homologous region that is identical in 20 of 21 residues and in which the predicted stem-loop structure is not affected and the motif defined by SELEX is conserved. This suggests that an impor- tant function is mediated by this region. Since L22 is also well conserved through evolution (1, 3), this function might be the interaction of the 28S ribosomal RNA with L22 protein. Third, this region perfectly matches the criteria that were shown to allow tight binding to L22 in the conventional SELEX exper- iment. Fourth, L22 is found in the large subunit of ribosomes, consistent with the prediction that it might interact directly with 28S ribosomal RNA in vivo. Finally, the associations of L22 with EBER1 or the ribosome are mutually exclusive (25). This suggests that at least one important contact between L22 and the rest of the ribosome may be formed through an RNA structure that has features similar to those of EBER1, resulting in the competition of EBER1 with a ribosomal RNA to bind L22.
In addition to cellobiose, other biological molecules have been used as targets for SELEX experiments. The first to be used was ATP (39). ATP is directly involved in the of energy of living systems (63). This selection was carried out by using ATP immobilized on a column matrix. RNA was passed over the column and binding species eluted by passing an excess of “free” ATP over the column. Following the selection, analysis of the cloned sequences showed that while highly divergent, all contained an 11 nucleotide stretch that folded into the same secondary structure. This structure was deduced to be a hairpin loop with an internal, asymmetric purine rich bulge. Once again SELEX was used to show that the four bases present in RNA were capable of recognizing small organic molecules, relatively simple chemical structures in biologically relevant molecules (Table 1.3).
Telomerase is a ribonucleoprotein enzyme that is responsible for the replication of telomeres at the ends of eukaryotic chromosomes (Cech 2000; Bryan and Cech 1999; Harley 2002; Collins and Mitchell 2002). In normal human cells there is little or no telomerase activity, whereas in 80% to 90% of tumor cells there is active enzyme (Neidle and Parkinson 2002). The importance of telomerase in cancer is highlighted by its role in the first reported oncogenic transformation of human cells caused by defined genetic changes (Hahn et al. 1999); expression of the human telomerase reverse transcriptase protein (hTERT) in combination with two oncogenes converts normal human cells to tumor cells. This and other work has led to the notion that inhibitors of telomerase may represent general antitumor therapeutics. The work in this thesis represents our progress in (1) understanding telomerase via a biochemical study of an important RNA-RNA interaction in telomerase and (2) performing in vitroselection against telomerase to isolate peptides that will lead to the development of anticancer therapies.
H epatitis C virus (HCV) is a positive-strand RNA virus encod- ing a polyprotein that is cleaved into 10 proteins, including three structural proteins (core, E1, and E2), a putative ion channel (p7), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (42). Propelled by the development of both subgenomic replicons (8, 31) and a cell culture-based infection system (HCVcc) (9, 29, 49, 54), research has garnered a wealth of information on the role of each of these proteins in the life cycle of the virus. The structural proteins and p7 are needed for virion assembly but not RNA replication, whereas the nonstructural pro- teins are probably involved in both replication and infectious vi- rus production. In addition to the requisite protease and polymer- ase activities, HCV also encodes proteins that perform specialized functions, such as deforming the membranes (to generate a mi- croenvironment for RNA replication) or scaffolding (to bring the appropriate proteins together for virion assembly) (14, 24, 37, 41). HCV infects ⬃3% of the world’s population and is a major risk factor for liver cirrhosis and hepatocellular carcinoma (5, 47). Current treatment comprises pegylated interferon (IFN), the nu- cleoside analog ribavirin, and recently approved direct-acting an- tivirals (DAAs) that inhibit the HCV NS3 protease. Despite the success of this triple therapy, drug resistance against the DAAs develops quickly (38), presumably because of both the error- prone nature of the viral polymerase (NS5B) and the high repli- cation rate in vivo. Host-targeting agents (HTAs) represent an alternative to DAAs, with an expected high genetic barrier to re- sistance because of the uncoupling of the selection pressure (on the virus) and the direct drug target (from the host). The leading HTAs that have progressed into clinical trials for HCV treatment are nonimmunosuppressive derivatives of cyclosporine (CsA), such as alisporivir (ALV; also known as DEB-025) (17), SCY-635 (23), and NIM-811 (27). These compounds inhibit the host pro- teins cyclophilins, primarily cyclophilin A (CyPA) (52), to block HCV replication and are collectively called cyclophilin inhibitors
Most functions of the NC protein involve nonspecific RNA interaction and do not require the presence of zinc fingers (9, 28). However, selection of virus-specific RNA containing the packaging site PSI from the pool of cellular RNAs has been shown to require an intact first zinc finger in vivo (10, 12, 21) and in vitro (8). The HIV Gag or NC proteins have been shown in gel shift studies to bind to PSI-containing RNA indepen- dently of the presence of the zinc fingers (3). The NC protein exhibits two clusters of basic amino acid residues which are characteristic for several RNA-binding proteins, such as Tat or Rev (20). In the case of NC proteins, this cluster is bipartite, flanking the first zinc finger. Deletions of short sequences con- taining these basic residues lead to a complete loss of nucleic acid binding activity in vitro (9). Furthermore, spumaviruses, retrotransposons, and copia-like retroelements contain basic amino acids but no zinc finger, supporting the importance of these sequences for RNAbinding (6). This study addresses questions concerning the function of the individual basic amino acid residues in RNAbinding in vitro.
The oligonucleotide pool used can be dsDNAt^], which can be amplified directly by PGR, ssDNAt^] or RNAM , which must then be converted to DNA using reverse transcriptase, amplified using PGR and transcribed back into RNA for another round of selection. To date most of the work done using this technique has focused on RNA. The design of the oligonucleotide pool is an important aspect to consider when attempting in vitroselection experiments. The probe must contain a randomised section that is usually created during standard phosphoroamidite synthesis by addition of equimolar amounts of the four activated nucleosides, but can be synthesised in other ways (e.g. from genomic digestiont^]). The random section is sandwiched between region of known sequences that enable the synthesis of complementary primers required for PGR, transcription and sequencing and to provide two restriction endonuclease sites to facilitate cloning. When using dsDNA, terminal GG rich regions are sometimes included in order to increase the stability of the duplexes, especially when using short oligonucleotides.
sequencing of randomly picked phagemid clones (Table 1). This 12-mer peptide-presenting phagemid library was selected in vitro against C2C12 mouse myoblasts as described earlier (2, 27) with several modifications. To reduce the number of promiscuous peptides that were selected against C2C12 myo- blasts, a clearing strategy was applied in which the phage were first adsorbed onto nontarget cells before the supernatant of this reaction was transferred to the target muscle cells. After five rounds of selection, 10 phage clones were picked and se- quenced to assess the repertoire of selected peptides (Table 2). A total of 30% of the clones were identical to the 12.51 se- quence, 20% were identical to 12.52 sequence, and 1 clone displayed the 12.53 sequence. The remaining clones either had a frameshift, truncation, or deletion in the sequence similar to that frequently observed when phagemid libraries were se- lected (26). Nevertheless, observation of the same peptide in multiple clones indicated that these peptides were likely se- lected for their cell-binding abilities (2, 27). BLAST of the 12.51, 12.52, and 12.53 sequences for similar proteins did not generate any matches with known proteins. These observations are consistent with our previous work with long peptide librar- FIG. 1. Phage binding of HI-RGD on an integrin-expressing cell
Analysis of a number of clones from every round (some 50 clones were analyzed) showed that many of the cDNA clones represented RNA sequences with a structure similar to that of SL1 but that many had no similarity to SL1. Those RNAs that did not have similarity to SL1 did not bind to viral particles in the gel shift assay. The binding to viral particles of T7 plus- strand RNAs from a selection of clones is shown in Fig. 3. All of the clones which failed to show any binding (e.g., 8-7, 8-13, 8-14, and 8-19) lacked the consensus secondary and primary structure (see below). Only those clones that had the consen- sus structure showed significant binding. Even at the eighth round of selection, nearly 50% of the clones isolated did not represent binding sequences. We have no ready explanation for this result. It may be that the gel electrophoretic separation of bound and unbound RNA was inadequate. Adding excess competitor AS transcript or excess noncompeting RNA to the binding reaction during selection had little effect on the clones isolated. A summary of the sequences of binding RNAs is shown in Table 1.
Another major advantage of using RNA to mediate the formation of materials is the ability to employ an in vitroselection approach (Scheme I1). 49 Nucleotide sequences that catalyze certain reactions or specifically bind to molecular targets can be selected from synthetic combinatorial RNA libraries. The libraries provide huge complexities of nucleotide sequence and structure (typically in the order of 10 14 ). During the in vitroselection process a pool of RNA sequences that are completely randomized at specific positions is subjected to selection pressure for catalysis or binding. The functional RNA's are selected during the partitioning step. The partitioning in the selection of RNA sequences can be designed to take advantage of binding constant or catalytic reactivity. Selected active sequences are reverse transcribed and amplified as dsDNA that is used for subsequent in vitro transcription. This newly transcribed RNA pool is enriched in the functional sequences and is then subjected to selection in the next cycle. Multiple rounds of enrichment result in the exponential increase of the active RNA species until they dominate the population of the sequences. This methodology assumes that the random pool of RNA sequences contains species performing a desired function. The in vitroselection approach has been successfully utilized to isolate a variety of RNA aptamers for a number of proteins and other biological receptors, as well as to small organic molecules. 50-54 Aptamers are nucleic acids that specifically bind to a target
Understanding viral proteinase specificity is of key impor- tance, not only for basic research, but also for designing anti- viral drugs that block proteinase activity. Potent inhibitors of cellular and viral proteases based on synthetic peptide ana- logues have been developed (18, 24, 32). Usually, the scissile dipeptide in these pseudosubstrates is replaced by a nonhydro- lyzable bond (7). In contrast, peptides used here to block poliovirus 2A pro activity in vitro lack a nonscissile Tyr-Gly pair.
the in vitro and in vivo selective aggregate binding of OCT coupled PEGylated liposomal nanoparticles radiolabeled with indium-111. The OCT derivative was synthesized by cross-linking of the S-acetyl-mercaptopropionic acid peptide with Mal-DSPE-PEG2000. Liposomes were obtained by mixing the OCT derivative with adequate amounts of palmitoyl oleoyl-phosphatidylcholine (POPC), lyso-stearyl- phosphatidylglycerol (Lyso-PG), distearyl- phosphatidylcholine–polyethyleneglycol-2000 (DSPE- PEG2000), and dimyristoyl phosphoethanolamine-DTPA (DMPE-DTPA) in a molar ratio of 0.1:11:7.5:0.9:2, respectively. Targeting properties of the OCT labeled lipo- somes were evaluated in vitro on rat pancreatic tumor cells (AR42J), demonstrating specific binding and IC 50 values in the low nanomolar range. Unfortunately only moderate uptake was observed when in vivo experiments were per- formed in animals; this may be explained by the limited and slow accessibility of target receptors on tumor cells by large constructs such as these, compared to small peptides that show much more rapid diffusion and binding to the receptors and cellular internalization.
to the 39 end of minus-strand sat-RNA C, and C59 (59-GGGATAACTAAG GGTTTCA-39), which is homologous to the 59 end of minus-strand sat-RNA C, were used to amplify either mutant or wild-type sat-RNA C cDNA by PCR, as previously described (39). For preparation of plus-strand templates, oligode- oxynucleotides T7C59 (59-GTAATACGACTCACTATAGGGATAACTAAGG G-39), which contained the T7 RNA polymerase promoter and 13 bases homol- ogous to the 5 9 end of plus-strand sat-RNA C, and C3 9 (5 9 -GGGCAGGCCC CCCGTCCGA-3 9 ), which is complementary to the 3 9 end of plus-strand sat- RNA C, were used for the PCR amplification. PCR products were cloned into the SmaI site of pUC19. Following SmaI digestion of the plasmid, plus- or minus-strand templates containing the exact 3 9 and 5 9 sequences were synthe- sized by using T7 RNA polymerase (23). After phenol-chloroform extraction and ethanol precipitation, the RNA concentration was determined by measuring the optical density at 260 nm of the in vitro transcription reaction mix and then subtracting the value contributed by the DNA template. Since the DNA is not a template for the RdRp (39), it was not removed from the DNA-RNA mixture. Construction of mutant sat-RNA C. CAM, CAMSL22, CAM260, CAM628, CAM182, CAM820, CAMP, CAM456, CAMX, CAM177, and CAM118 were described by Cascone et al. (10). CAMd14 was described by Cascone (8). CSN was described by Simon et al. (37). CNL5 was described by Carpenter et al. (5). pCd3 9 8 was described by Song and Simon (39). To prepare sat-RNA C cDNA that would generate minus-strand sat-RNA templates with 5 9 deletions of 5 or 22 bases or with four altered bases near the 5 9 end (DM1), oligodeoxynucleotides that contained the T7 RNA polymerase promoter and sequence homologous to the 5 9 end of sat-RNA C, including the deletions or base alterations, were synthesized and used in the PCRs described above. To introduce mutations into the region immediately upstream of the template-product junction, site-directed mutagenesis was performed as described by Kunkel et al. (21) with a partially degenerate oligonucleotide [5 9 -AACCTGGCT(A/C)(A/G)(A/G)(A/G)GGGA (G/T)TCAAAAGAATCC-3 9 ].
Background: Solid-bindingpeptides (SBPs) bind strongly to a diverse range of solid materials without the need for any chemical reactions. They have been used mainly for the functionalisation of nanomaterials but little is known about their use for the immobilisation of thermostable enzymes and their feasibility in industrial-scale biocatalysis. Results: A silica-binding SBP sequence was fused genetically to three thermostable hemicellulases. The resulting enzymes were active after fusion and exhibited identical pH and temperature optima but differing thermostabili- ties when compared to their corresponding unmodified enzymes. The silica-binding peptide mediated the efficient immobilisation of each enzyme onto zeolite, demonstrating the construction of single enzyme biocatalytic modules. Cross-linked enzyme aggregates (CLEAs) of enzyme preparations either with or without zeolite immobilisation dis- played greater activity retention during enzyme recycling than those of free enzymes (without silica-binding peptide) or zeolite-bound enzymes without any crosslinking. CLEA preparations comprising all three enzymes simultaneously immobilised onto zeolite enabled the formation of multiple enzyme biocatalytic modules which were shown to degrade several hemicellulosic substrates.
TACGACTCACTATAAGCAAAGCAGGGTTA-39, corresponding to the 59 and 39 termini of segment, 5, respectively. The underlined portions of the primers indicate the sequence of the T7 promoter that was incorporated into the final PCR product. The bases in boldface indicate the mutations introduced into the T7 promoter, which would direct transcription initiation with a sequence corre- sponding to the 59 terminus of the influenza virus genome. The truncated NP FIG. 1. (A) A deleted NP gene (152 nt long) of vRNA sense was used as a template in a transcription reaction mixture containing RNP-derived polymerase from which endogenous influenza virus RNA had been depleted by treatment of isolated RNPs with micrococcal nuclease, as described in Materials and Meth- ods. Radiolabeled RNA transcripts from the reaction were resolved in a 6% polyacrylamide–7 M urea gel and visualized by autoradiography. Lane M, 152-nt RNA marker; lane 1, transcription products of MT-RNPs (0.6 mg) in the pres- ence of 0.4 mM ApG; lane 2, the same as lane 1 but without ApG; lane 3, 1 mg of truncated vRNA incubated with 0.6 mg of MT-RNPs in a transcription reac- tion mixture primed with 0.4 mM ApG; lane 4, a transcription reaction the same as that in lane 3 but primed with 1 mM AlMV4. (B) A cRNA template (152 nt long) was assayed in a transcription reaction as described above. Lane M, RNA marker; lane 1, transcription products of MT-RNPs (0.6 m g) in the presence of 0.4 mM ApG; lane 2, the same as lane 1 but without ApG. In lanes 3, 4, and 5, 1 m g of truncated cRNA was incubated with 0.6 m g of MT-RNPs in a transcrip- tion reaction mixture containing ApG, AlMV4, or a 12-nt-long oligoribonucle- otide (Oligoribo) complementary to the 3 9 terminus of the cRNA.
Design of an effective PDZ-binding peptide included considerations of ability to access an intracellular target and stability in both extra-and intra-cellular environments. Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across cell membrane. Natural CPPs occur in basic peptide sequences (10-14aa) such as that derived from HIV-encoded TAT transactivator protein, or the penetratin protein (16aa) derived from the antennapedia protein of Drosophilia. Synthetic arginine-rich peptides also function as CPPs (Futaki et al., 2001). We chose to incorporate a hexa-D- arginine sequence as it has previously been shown that cell penetrating peptides are stabilized by D-residues without loss of cell penetrating activity (Futaki et al, 2001). When linked N-terminal to biotinylated cyclic PDZ- bindingpeptides, the hexa-DArg sequence was highly effective at localizing its cargo throughout SHSY-5Y cells (Figure 2), whereas a control peptide lacking 6xDArg (figure 2F) was not visualized inside cells after the same incubation conditions.