用ribozyme抑制增殖细胞核抗原的表达对HaLa细胞增殖的影响

增殖细胞核抗原(PCNA)是DNA聚合酶δ的辅助蛋白,它是细胞染色体DNA复制所必需的。人工设计的ribozyme具有可特异地切割PCNA mRNA的性质,将此ribozyme的自修剪体内表达质粒导入HeLa细胞,从细胞总RNA中分离相应部分能在体外切割靶RNA片段,证明此表达质粒在细胞内能表达出有活性的ribozyme分子。与对照相比,导入ribo-zyme表达质粒的HeLa细胞进入S期的时间从12 h推迟到20 h,而突变ribozyme的对照表明反义抑制对细胞进入S期的影响较小(推迟到15 h)。证明该ribozyme能有效抑制He-La细胞DNA复制,同时亦证明PCNA对于细胞DNA复制及细胞周期进程的重要性。     

The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.

The addition of the hepatitis delta virus genomic ribozyme to the 3' end sequence of a Sendai virus defective interfering RNA (DI-H4) allowed the reproducible and efficient replication of this RNA by the viral functions expressed from cloned genes when the DI RNA was synthesized from plasmid. Limited nucleotide additions or deletions (+7 to -7 nucleotides) in the DI RNA sequence were then made at five different sites, and the different RNA derivatives were tested for their abilities to replicate. Efficient replication was observed only when the total nucleotide number was conserved, regardless of the modifications, or when the addition of a total of 6 nucleotides was made. The replicated RNAs were shown to be properly enveloped into virus particles. It is concluded that, to form a proper template for efficient replication, the Sendai virus RNA must contain a total number of nucleotides which is a multiple of 6. This was interpreted as the need for the nucleocapsid protein to contact exactly 6 nucleotides.     

The prebiotic evolutionary advantage of transferring genetic information from RNA to DNA

In the early 'RNA world' stage of life, RNA stored genetic information and catalyzed chemical reactions. However, the RNA world eventually gave rise to the DNA-RNA-protein world, and this transition included the 'genetic takeover' of information storage by DNA. We investigated evolutionary advantages for using DNA as the genetic material. The error rate of replication imposes a fundamental limit on the amount of information that can be stored in the genome, as mutations degrade information. We compared misincorporation rates of RNA and DNA in experimental non-enzymatic polymerization and calculated the lowest possible error rates from a thermodynamic model. Both analyses found that RNA replication was intrinsically error-prone compared to DNA, suggesting that total genomic information could increase after the transition to DNA. Analysis of the transitional RNA/DNA hybrid duplexes showed that copying RNA into DNA had similar fidelity to RNA replication, so information could be maintained during the genetic takeover. However, copying DNA into RNA was very error-prone, suggesting that attempts to return to the RNA world would result in a considerable loss of information. Therefore, the genetic takeover may have been driven by a combination of increased chemical stability, increased genome size and irreversibility.     

Combinatorial minimization and secondary structure determination of a nucleotide synthase ribozyme

We previously isolated from random sequences ribozymes able to form a glycosidic linkage between a ribose sugar and 4-thiouracil in a reaction that mimics protein-catalyzed nucleotide synthesis. Here we report on two serial in vitro selection experiments that defined the core motif of one of the nucleotide synthase ribozymes and provided improved versions of this ribozyme. The first selection experiment started from a degenerate sequence pool based on the previously isolated sequence and used a selection-amplification protocol that allowed the sequence requirements at the 3' terminus of the ribozyme to be interrogated. Comparing the active sequences identified in this experiment revealed the complicated secondary structure of the nucleotide synthase ribozyme. A second selection was then performed to remove nonessential sequence from the ribozyme. This selection started with a pool with variation introduced in both the sequence and the length of the nonconserved loops and joining regions. This pool was generated using a partial reblocking/deblocking strategy on a DNA synthesizer, allowing the combinatorial synthesis of both point deletions and point substitutions. The consensus ribozyme motif that emerged was an approximately 71 nt pseudoknot structure with five stems and two important joining segments. Comparative sequence analysis and a cross-linking experiment point to the probable location of nucleotide synthesis. The prototype isolate from the second selection was nearly 35 times more efficient than the initial isolate and at least 10(8) times more efficient than an upper limit of an as-yet undetectable uncatalyzed reaction, supporting the idea that RNA-catalyzed nucleotide synthesis might have been important in an RNA world.     

Errors and alternatives in prebiotic replication and catalysis

The work on nonenzymatic nucleic acid replication performed by Leslie Orgel and co-workers over the last four decades, now extended by work on artificial selection of RNA aptamers and ribozymes, is generating some pessimism concerning the 'naked gene' theories of the origin of life. It is suggested here that the low probability of finding RNA aptamers and ribozymes within pools of random sequences is not as disquieting as the poor gain in efficiency obtained with increases in information content. As acknowledged by Orgel and many other authors, primitive RNA replication and catalysis must have occurred within already complex and dynamic environments. I, thus, propose to pay attention to a number of possibilities that bridge the gap between 'naked gene' theories, on one side, and metabolic theories in which complex systems self-propagate by growth and fragmentation, on the other side. For instance, one can de-emphasize nucleotide-by-nucleotide replication leading to long informational polymers, and view instead long random polymers as storage devices, from which shorter oligomers are excised. Catalytic tasks would be mainly performed by complexes associating two or more oligomers belonging to the same or to different chemical families. It is proposed that the problems of stability, binding affinity, reactivity, and specificity could be easier to handle by heterogeneous complexes of short oligomers than by long, single-stranded polymers. Finally, I point out that replication errors in a primitive replication context should include incorporations of alternative nucleotides with interesting, chemically reactive groups. In this way, an RNA sequence could be at the same time an inert sequence when copied without error, and a ribozyme, when a chemically reactive nucleotide is inadvertently introduced during replication.     

An Interpretive Review of the Origin of Life Research

Life appears to be a natural property of matter, but the problem of its origin only arose after early scientists refuted continuous spontaneous generation. There is no chance of life arising ‘all at once’, we need the standard scientific incremental explanation with large numbers of small steps, an approach used in both physical and evolutionary sciences. The necessity for considering both theoretical and experimental approaches is emphasized. After describing basic principles that are available (including the Darwin-Eigen cycle), the search for origins is considered under four main themes. These are the RNA-world hypothesis; potential intermediates between an RNA-world and a modern world via the evolution of protein synthesis and then of DNA; possible alternatives to an RNA-world; and finally the earliest stages from the simple prebiotic systems to RNA. The triplicase/proto-ribosome theory for the origin of the ribosome is discussed where triples of nucleotides are added to a replicating RNA, with the origin of a triplet code well-before protein synthesis begins. The length of the code is suggested to arise from the early development of a ratchet mechanism that overcomes the problem of continued processivity of an RNA-based RNA-polymerase. It is probable that there were precursor stages to RNA with simpler sugars, or just two nucleotides, but we do not yet know of any better alternatives to RNA that were likely to arise naturally. For prebiotic stages (before RNA) a flow-reactor model is suggested to solve metabolism, energy gradients, and compartmentation simultaneously – thus the intense interest in some form of flow reactor. If an autocatalytic cycle could arise in such a system we would be major steps ahead. The most likely physical conditions for the origin of life require further clarification and it is still unclear whether the origin of life is more of an entropy (information) problem (and therefore high temperatures would be detrimental), rather than a kinetic problem (where high temperatures may be advantageous).     

Co-evolution of the genetic code and ribozyme replication

The origin of translation has stimulated much discussion since the basic processes involved were deciphered during the 1960s and 1970s. One strand of thought suggested that the process originated from RNA replication in the RNA world (Weiner & Maizels, 1987, 1994). In this paper I seek to extend this model. The mRNA originates as a replication intermediate of minus-strand ribozyme replication and thus contains all the genetic information contained in both the ribozyme portion and the putative tRNA-like portion of the RNA molecule. Qualitatively, this is similar to the model for the origin of chromosomes (Szathmary & Maynard-Smith, 1993, Maynard-Smith & Szathmary, 1993). This model explicitly describes the evolution of early chromosomes and the role replication played in generating the modern mRNA. Moreover, by pursuing this model, the START and STOP codons were derived and their original function with regard to the primitive 23S ribosomal RNA is suggested. Co-evolution of the genetic code (Wong, 1975) is also contained within the model. Lastly, I address some of the benefits and costs that the process may have for the organism in the context of autotrophy in the RNA world.     

In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

In vitro selection and characterization of cellulose-binding RNA aptamers using isothermal amplification

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.     

Evolutionary origins and directed evolution of RNA

In vitro selection experiments show first and foremost that it is possible that functional nucleic acids can arise from random sequence libraries. Indeed, even simple sequence and structural motifs can prove to be robust binding species and catalysts, indicating that it may have been possible to transition from even the earliest self-replicators to a nascent, RNA-catalyzed metabolism. Because of the diversity of aptamers and ribozymes that can be selected, it is possible to construct a 'fossil record' of the evolution of the RNA world, with in vitro selected catalysts filling in as doppelgangers for molecules long gone. In this way a plausible pathway from simple oligonucleotide replicators to genomic polymerases can be imagined, as can a pathway from basal ribozyme activities to the ribosome. Most importantly, though, in vitro selection experiments can give a true and quantitative idea of the likelihood that these scenarios could have played out in the RNA world. Simple binding species and catalysts could have evolved into other structures and functions. As replicating sequences grew longer, new, more complex functions or faster catalytic activities could have been accessed. Some activities may have been isolated in sequence space, but others could have been approached along large, interconnected neutral networks. As the number, type, and length of ribozymes increased, RNA genomes would have evolved and eventually there would have been no area in a fitness landscape that would have been inaccessible. Self-replication would have inexorably led to life.     

Fine mapping of a cis-acting sequence element in yellow fever virus RNA that is required for RNA replication and cyclization

We present fine mapping of a cis-acting nucleotide sequence found in the 5' region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3' region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5' region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquito-borne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.     

Prebiotic chemistry and the origin of the RNA world

The demonstration that ribosomal peptide synthesis is a ribozyme-catalyzed reaction makes it almost certain that there was once an RNA World. The central problem for origin-of-life studies, therefore, is to understand how a protein-free RNA World became established on the primitive Earth. We first review the literature on the prebiotic synthesis of the nucleotides, the nonenzymatic synthesis and copying of polynucleotides, and the selection of ribozyme catalysts of a kind that might have facilitated polynucleotide replication. This leads to a brief outline of the Molecular Biologists' Dream, an optimistic scenario for the origin of the RNA World. In the second part of the review we point out the many unresolved problems presented by the Molecular Biologists' Dream. This in turn leads to a discussion of genetic systems simpler than RNA that might have "invented" RNA. Finally, we review studies of prebiotic membrane formation.     

The two ages of the RNA world, and the transition to the DNA world: a story of viruses and cells

Most evolutionists agree to consider that our present RNA/DNA/protein world has originated from a simpler world in which RNA played both the role of catalyst and genetic material. Recent findings from structural studies and comparative genomics now allow to get a clearer picture of this transition. These data suggest that evolution occurred in several steps, first from an RNA to an RNA/protein world (defining two ages of the RNA world) and finally to the present world based on DNA. The DNA world itself probably originated in two steps, first the U-DNA world, following the invention of ribonucleotide reductase, and later on the T-DNA world, with the independent invention of at least two thymidylate synthases. Recently, several authors have suggested that evolution from the RNA world up to the Last Universal Cellular Ancestor (LUCA) could have occurred before the invention of cells. On the contrary, I argue here that evolution of the RNA world taken place in a framework of competing cells and viruses (preys, predators and symbionts). I focus on the RNA-to-DNA transition and expand my previous hypothesis that viruses played a critical role in the emergence of DNA. The hypothesis that DNA and associated mechanisms (replication, repair, recombination) first evolved and diversified in a world of DNA viruses infecting RNA cells readily explains the existence of viral-encoded DNA transaction proteins without cellular homologues. It also potentially explains puzzling observations from comparative genomic, such as the existence of two non-homologous DNA replication machineries in the cellular world. I suggest here a specific scenario for the transfer of DNA from viruses to cells and briefly explore the intriguing possibility that several independent transfers of this kind produced the two cell types (prokaryote/eukaryote) and the three cellular domains presently known (Archaea, Bacteria and Eukarya).     

Reovirus protein sigmaNS binds in multiple copies to single-stranded RNA and shares properties with single-stranded DNA binding proteins

Reovirus nonstructural protein sigmaNS interacts with reovirus plus-strand RNAs in infected cells, but little is known about the nature of those interactions or their roles in viral replication. In this study, a recombinant form of sigmaNS was analyzed for in vitro binding to nucleic acids using gel mobility shift assays. Multiple units of sigmaNS bound to single-stranded RNA molecules with positive cooperativity and with each unit covering about 25 nucleotides at saturation. The sigmaNS protein did not bind preferentially to reovirus RNA over nonreovirus RNA in competition experiments but did bind preferentially to single-stranded over double-stranded nucleic acids and with a slight preference for RNA over DNA. In addition, sigmaNS bound to single-stranded RNA to which a 19-base DNA oligonucleotide was hybridized at either end or near the middle. When present in saturative amounts, sigmaNS displaced this oligonucleotide from the partial duplex. The strand displacement activity did not require ATP hydrolysis and was inhibited by MgCl(2), distinguishing it from a classical ATP-dependent helicase. These properties of sigmaNS are similar to those of single-stranded DNA binding proteins that are known to participate in genomic DNA replication, suggesting a related role for sigmaNS in replication of the reovirus RNA genome.     

Launching of the yeast 20 s RNA narnavirus by expressing the genomic or antigenomic viral RNA in vivo

20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3'-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3'-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5'-GGGGC... GCCCC-OH). The fifth G from the 3'-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3'-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3'-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3'-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3'-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons.     

Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3'-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world.