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At least 19 records

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NASA NTRS2001

Substitution of Ribonucleotides in the T7 RNA Polymerase Promoter Element

A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the non-template and template strands of the promoter. These residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and P-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing its promoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that employ T7 RNA polymerase for in vitro transcription.

McGinness, Kathleen E.↗

NASA NTRS1992

Oligomerization reactions of ribonucleotides - The reaction of the 5'-phosphorimidazolide of nucleosides on montmorillonite and other minerals

The reaction of ImpA in the presence of Na(+)-montmorillonite 22A or Na(+)-Volclay in aqueous, pH 8 solution gives a 50-60 percent yield of dimers and trimers (pA)2 and (pA)3. The ratio of 3',5'-phosphodiester bond formation is twice as great as 2',5'-bond formation. The reaction requires the presence of Mg2+ and is inhibited by 0.4 M imidazole. N-methylimidazole enhances the rate of the reaction but does not cause major changes in yield or product composition. Higher yields were obtained when Li+- or Ca2+-montmorillonites were used in place of Na+-montmorillonite. Little or no phosphodiester bond formation was observed with Mg2+- or Al3+-montmorillonite. Montmorillonites other than 22A and Volclay exhibited litle or no catalysis. In addittion, little or no catalysis was exhibited in ferrugenous smectite, nontronite, allophane, imogolite or sepiolite. Oligomers were also formed by the reaction of ImpG, 2-methylImpG, ImpC and ImpU in the presence of Na+-montmorillonite. The pyrimidine nucleotides gave significantly lower yields of oligomers.

Ferris, James P.↗

NASA NTRS1993

Oligomerization reactions of ribonucleotides - The reaction of the 5'-phosphorimidazolide of adenosine with diadenosine pyrophosphate on montmorillonite and other minerals

The overall regiospecificity of the 3',5'-phosphodiester bond formation is shown to be increased from 64 to 83 percent when a 9:1 ratio of ImpA to A5'ppA instead of ImpA alone is reacted in the presence of Na(+)-montmorillonite. The major factor in the regiospecificity increase is the formation of 3',5'-linked A5'ppA3'pA and A5'pp-A3'pA3'pA. The results obtained show that ImpA reacts more readily with A5'ppA than it does with another ImpA molecule and that A5'ppA plays a central role in directing the formation of 3',5'-phosphodiester bonds.

Ferris, James P.↗

NASA NTRS2002

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Ordoukhanian, Phillip↗

NASA NTRS1992

On the early emergence of reverse transcription: theoretical basis and experimental evidence

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.

Non-NASA Center↗

NASA NTRS2015

Life in the Clouds of Venus? An Experimental Synthetic Biology Approach

The surface of Venus constitutes the most hellish and biologically inhospitable planetary surface in our solar system, boasting a pH of 0, blistering winds that can melt lead, and pressures of 60 atm. However, during the earlier years of the solar system, without the runaway greenhouse effect that has plagued the planet, Venus potentially housed oceans and perhaps even life. There is a possibility that microbes could have retreated into hospitable niches in the atmosphere, as suggested by Carl Sagan as early as 1967 [1]. For example, 50 km above the raging hell of the Venusian surface, exists a relatively temperate environment that might serve as reservoir for life. This astrobiology project seeks to explore life at the extremes and to theorize whether microbial communities could not only survive but also reproduce in the Venusian atmosphere. Specifically, we ask: are aerosols viable microbial environments? But before we can test for life in the clouds, we have to develop a proper reporter to visualize cell growth in situ. For this purpose, we aimed to develop cell-growth dependent reporters to serve as remote biosensors for cell growth. We developed two using the polA promoter, a DNA-replication dependent promoter, and nrd operon promoter, a cell-cycle dependent promoter. Using these cell-growth reporters, the next step is to aerosolize microbes expressing these reporters in a suspension chamber adapted from a Millikan Drop Apparatus to assay reproduction in an aerosolized environment. Better yet is to test the reproduction of microbes in a microgravity regime such as on ISS.Approach: We engineered two cell-cycle dependent genetic reporters. One was the polA promoter which codes for DNA Polymerase I, a gene active in DNA replication [2]. The other was the nrdP. The activation of ribonucleotide reductase reduces ribonucleotides into deoxyribonucleotides and is involved in the bacterial cell cycle [3]. This promoter began activation during the initiation of DNA replication and is cell-cycle dependent [4]. These promoters were fused to a GFP reporter, transformed into E. coli. The constructs were deposited in the iGEM registry as K847210: Escherichia coli DNA-replication dependent polA promoter K847211: Escherichia coli cell-division dependent nrd promoter Results: Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrdP-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polAP-E0840 construct (which uses mut3b GFP) exhibited low expression; while fluorescence was visible under the microscope, the signal was too weak for the camera to recognize. The polA promoter was therefore digest-ed with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness [5] Fluoresence time course data demonsrated that the genes were induced in a cell cycle dependant manner [6]. Our assays via microscopy and the bulk assay shows that our promoters are functional as cell cycle reporters.Conclusions: The application of such tools are widespread and not limited to astrobiology; nrdP could be used to determine doubling times empirically and could possibly extrapolate DNA content from intensity of signals expressed by polAP. However, we are pri-marily interested in its use in astrobiology.

Escherichia↗

NASA NTRS1973

Prebiotic activation processes.

Questions regarding the combination of amino acids and ribonucleotides to polypeptides and polynucleotides are investigated. Each of the reactions considered occurs in the solid state in plausible prebiotic conditions. Together they provide the basis for a unified scheme of amino acid and nucleotide activation. Urea, imidazole and Mg(++) are essential catalytic components of the reaction mixtures. However, these compounds could probably be replaced by other organic molecules.

Lohrmann, R.↗

NASA NTRS1981

Reduction of thionucleosides - A prebiotic pathway to deoxyribonucleosides

A mechanism is proposed for the prebiotic synthesis of deoxyribonucleotides and possible nucleic acid analogs from ribonucleotides by a pathway involving 2'-thio-2'-deoxyribonucleosides. The mechanism is supported by laboratory experiments in which 2'-thio-2'-deoxycytidine was synthesized from anhydro arabinosyl cytosine in dithiophosphate and CS2. The subsequent reduction of the thio-analogs has been achieved with ferrous ion, and photochemically. It is noted that the proposed pathway for prebiotic deoxyribonucleotide synthesis is in harmony with the Principle of Continuity, as both the proposed and present pathways rely on the reduction of a 2' functional group.

Patel, A. D.↗

NASA NTRS1982

Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

Wang, A. H.-J.↗

NASA NTRS1987

The case for an ancestral genetic system involving simple analogues of the nucleotides

The idea that the first living systems on earth were based on self-replicating RNA molecules has recently become popular as a result of the discovery of ribozymes. However, there are several major problems associated with the prebiotic synthesis of ribonucleotides. In addition, there is the newly recognized problem of enantiomeric cross-inhibition, whereby template-directed polymerization involving one enantiomer of RNA is inhibited strongly by the presence of the other enantiomer. Here, it is proposed that RNA was preceded in the evolution of life by a polymer constructed from flexible, acyclic, probably prochiral nucleotide analogues that were synthesized readily on the primitive earth. Several potentially prebiotic nucleotide analogues are considered in this context, and some of the consequences of this proposal are discussed.

Joyce, Gerald F.↗

NASA NTRS1989

Oligomerization reactions of deoxyribonucleotides on montmorillonite clay - The effect of mononucleotide structure on phosphodiester bond formation

The formation of oligomers from deoxynucleotides, catalyzed by Na(+)-montmorillonite, was investigated with special attention given to the effect of the monomer structure on the phosphodiester bond formation. It was found that adenine deoxynucleotides bind more strongly to montmorillonite than do the corresponding ribonucleotides and thymidine nucleotides. Tetramers of 2-prime-dpA were detected in the reaction of 2-prime-d-5-prime-AMP with a water-soluble carbodiimide EDAC in the presence of Na(+)-montmorillonite, illustrating the possible role of minerals in the formation of biopolymers on the primitive earth.

Ferris, James P.↗

NASA NTRS1995

The Relative Reactivity of Deoxyribose and Ribose: Did DNA Come Before RNA?

If it is assumed that there was a precursor to the ribose-phosphate backbone of RNA in the preRNA world (such as peptide nucleic acid), then the entry of various sugars into the genetic material may be related to the stability and non-enzymatic reactivity of the aldose. The rate of decomposition of 2-deoxyribose has been determined to be 1/3 that of ribose. In addition we have measured the amount of free aldehyde by H-1 and C-13 NMR and find that it has approximately 0.15% free aldehyde compared to 0.05% for ribose at 25 C. This suggests that deoxyribose would be significantly more reactive with early bases in the absence of enzymes. This is confirmed by urazole and deoxyribose reacting to form the deoxynucleoside 45 times faster as 25 C than urazole reacts with ribose to form the Ribonucleoside. Urazole is a potential precursor of uracil and is a plausible prebiotic compound which reacts with aldoses to form nucleosides. Thus the non-enzymatic reactivity of deoxyribose would favor its early use over ribose until enzymes could change the relative reactivities. Most of the reasons that RNA is presumed to have come before DNA are extrapolations back from contemporary metabolism (e.g. the abundance of ribose based coenzymes, the biosynthesis of histidine, deoxyribonucleotides are synthesized from ribonucleotides, etc.). It is very difficult to reconstruct biochemical pathways much before the last common ancestor, and it is even more difficult to do more than guess at the biochemistry of very early self-replicating systems. Thus we believe that these reasons are not compelling and that the non-enzymatic chemistry may be more important than enzymatic pathways for constructing the earliest of biochemical pathways. While the RNA world has been discussed at great length, there has not been an exploration of the transition out of the RNA world. We have constructed many possible schemes of genetic takeover events from preRNA to modern DNA, RNA, protein system which could generate the RNA metabolic fossils we see today.

Dworkin, Jason P.↗

NASA NTRS1992

The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or polymers with related peptide backbones were the dominant information macromolecules.

Nelson, Kevin E.↗

NASA NTRS2004

Did the Pre-RNA World Rest Upon DNA Molecules?

The isolation of a DNA sequence that catalyzes the ligation of oligodeoxynucleotides via the formation of 3' - 5' phosphodiester linkage significance in selection experiments has been reported. Ball recently used this to discuss the possibility that natural DNA molecules may have formed in the primitive Earth leading to the origin of life. As noted by Ferris and Usher, if metabolic pathways evolved backwards, it could be argued that the biosynthesis of 2-deoxyribose from ribose suggests that RNA came from DNA. As summarized elsewhere, there are several properties of deoxyribose which could be interpreted to support the possibility that DNA-like molecules arose prior to the RNA world. For example, 2-deoxyribose is slightly more soluble than ribose (which may have been an advantage in a drying pool scenario), may have been more reactive under possible prebiotic conditions (it forms a nucleoside approx. 150 times faster than ribose with the alternative base urazole at 25 C), while it decomposes in solution (approximately 2.6 times more slowly than ribose at 100 C). Other advantages of DNA over RNA are that it has one fewer chiral center, has a greater stability at the 8.2 pH value of the current oceans, and does not has the 2'5' and 3'5' ambiguity in polymerizations. Yet, there is strong molecular biological and biochemical evidence that RNA was featured in the biology well before the last common ancestor. The presence of sugar acids, including both ribo- and deoxysugar acids, in the 4.6 Ga old Murchison meteorite suggest that both may have been available in the primitive Earth, derived from the accretion of extraterrestrial sources and/or from endogenous processes involving formaldehyde and its derivatives. However, the abiotic synthesis of deoxyribose, ribose, and other sugars from glyceraldehyde and acetaldehyde under alkaline conditions is inefficient and unespecific. Although sugars are labile compounds, the role of cyanamide or borate minerals in the stabilization of the cyclic forms of ribose and other pentoses has recently been demonstrated. Nonetheless, the assumption either RNA or DNA was the first genetic material needs to be supplemented by laboratory models demonstrating that the prebiotic synthesis of activated beta-D-(deoxy)ribonucleotides and their polymers was feasible. As of today such evidence is lacking, and there is no convincing synthesis of any nucleotide, since all model experiments produce complex mixtures of products in which there is no preferential synthesis of chiral D-nucleotides. This strongly suggests that both DNA and RNA may have been preceded by pairing structures much simpler than extant nucleic acids. It is doubtful that DNA molecules, or indeed other (de0xy)ribofuranoid oligonucleotides formed the basis of these as yet undescribed pre-RNA worlds.

Lazcano, Antonio↗

NASA NTRS1999

Oligomerization of deoxyguanosine 5'-phosphoro-2-methylimidazolide on a polycytidylate template

The oligomerization of deoxyguanosine 5'-phosphoro-2-methylimidazolide on a polycytidylate template is much less efficient than the oligomerization of the corresponding activated ribonucleotide. Nonetheless oligomers containing up to eight nucleotide residues are detected. The products are 3'-5'-linked oligodeoxyribonucleotides capped at the 5'-terminus with a pyrophosphate-linked monomer.

NASA Discipline Exobiology↗

NASA NTRS1997

Information transfer from peptide nucleic acids to RNA by template-directed syntheses

Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.

NASA Discipline Exobiology↗

NASA NTRS2000

Nucleozymes

Nucleozymes containing ribonucleotides and deoxyribonucleotides or nucleic acid analogues are described herein. The nucleozymes have catalytic activity and are significantly more resistant to degradation than their all-RNA ribozyme counterparts. Also described are methods for preparing the nucleozymes along with methods of using nucleozymes, e.g., as therapeutic agents.

Usman, Nassim↗

NASA NTRS2004

Nucleozymes

Usman, Nassim↗