Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying.

2-Thiouridine (s2U) is a nucleobase modification that confers enhanced efficiency and fidelity both on modern tRNA codon translation and on nonenzymatic and ribozyme-catalyzed RNA copying. We have discovered an unusual base pair between two 2-thiouridines that stabilizes an RNA duplex to a degree that is comparable to that of a native A:U base pair. High-resolution crystal structures indicate similar base-pairing geometry and stacking interactions in duplexes containing s2U:s2U compared to those with U:U pairs. Notably, the C═O···H-N hydrogen bond in the U:U pair is replaced with a C═S···H-N hydrogen bond in the s2U:s2U base pair. The thermodynamic stability of the s2U:s2U base pair suggested that this self-pairing might lead to an increased error frequency during nonenzymatic RNA copying. However, competition experiments show that s2U:s2U base-pairing induces only a low level of misincorporation during nonenzymatic RNA template copying because the correct A:s2U base pair outcompetes the slightly weaker s2U:s2U base pair. In addition, even if an s2U is incorrectly incorporated, the addition of the next base is greatly hindered. This strong stalling effect would further increase the effective fidelity of nonenzymatic RNA copying with s2U. Our findings suggest that s2U may enhance the rate and extent of nonenzymatic copying with only a minimal cost in fidelity.

A Facile and General Tandem Oligonucleotide Synthesis Methodology for DNA and RNA.

Tandem oligonucleotide synthesis (TOS) is an attractive strategy to increase automated oligonucleotide synthesis efficiency. TOS is accomplished via the introduction of an immolative linker within a single sequence composed of multiple oligonucleotide fragments. Here, we report the use of a commercially available building block, typically utilized for the chemical phosphorylation of DNA/RNA oligomers, to perform TOS. We show that the 2,2'-sulfonyldiethylene linker is efficiently self-immolated during the standard deprotection of DNA and RNA and presents itself as a generalizable methodology for nucleic acid TOS. Furthermore, we show the utility of this methodology by assembling a model siRNA construct, and showcase a template-directed ligation pathway to incorporate phosphoramidate or pyrophosphate linkages within DNA oligomers.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Validation of oscillometric blood pressure measurement using a Datex S/5 Compact multiparameter monitor in anaesthetized adult dogs.

OBJECTIVE: To compare noninvasive (NIBP) with invasive blood pressure (IBP) measurements from a Datex S/5 Compact monitor in anaesthetized adult dogs, and to evaluate it according to the American College of Veterinary Internal Medicine (ACVIM) and the Association for the Advancement of Medical Instrumentation (AAMI) criteria. STUDY DESIGN: Prospective clinical study. ANIMALS: A group of 34 client-owned adult dogs. METHODS: Dogs were anaesthetized for different surgical procedures using different anaesthetic protocols. IBP was measured using a catheter placed in a dorsal pedal artery. A blood pressure cuff was placed over the contralateral dorsal pedal artery for NIBP measurement. Data were recorded using the Datex iCollect program, and paired readings were matched every 3 minutes for 60 minutes. Bland-Altman and error grid analyses were used to estimate the agreement between IBP and NIBP measurements, and its clinical significance, respectively. Data were reported as mean bias [lower, upper limits of agreement (LoA)]. RESULTS: The Datex S/5 monitor conformed to most ACVIM criteria. The correlation coefficient was less than 0.9 for systolic, diastolic, and mean arterial pressures (MAP). The best agreement between the noninvasive and invasive methods was observed for MAP, with LoA (-17 to 13 mmHg) and higher percentage of NIBP readings within 5 (55.6%), 10 (81.7%) and 20 (98.6%) mmHg of the IBP values. The Datex S/5 NIBP technology did not meet the AAMI validation criteria and less than 95% of the paired measurements were found within the green zone of the error grid analysis. CONCLUSIONS AND CLINICAL RELEVANCE: The Datex S/5 monitor conformed to most ACVIM criteria but not with the more rigorous AAMI standards. Despite good agreement between IBP and NIBP for MAP measurements, care must be taken when using this device to guide therapeutic interventions of blood pressure in anaesthetized healthy adult dogs.

Accuracy and trending capability of haemoglobin measurement by noninvasive pulse co-oximetry in anaesthetized horses.

OBJECTIVE: To assess the accuracy and trending capability of continuous measurement of haemoglobin concentration [Hb], haemoglobin oxygen saturation (SaO2) and oxygen content (CaO2) measured by the Masimo Radical-7 pulse co-oximeter in horses undergoing inhalational anaesthesia. STUDY DESIGN: Prospective observational clinical study. ANIMALS: A group of 23 anaesthetized adult horses. METHODS: In 23 healthy adult horses undergoing elective surgical procedures, paired measurements of pulse co-oximetry-based haemoglobin concentration (SpHb), SaO2 (SpO2), and CaO2 (SpOC) and simultaneous arterial blood samples were collected at multiple time points throughout anaesthesia. The arterial samples were analysed by a laboratory co-oximeter for total haemoglobin (tHb), SaO2 and manually calculated CaO2. Bland-Altman plots, linear regression analysis, error grid analysis, four-quadrant plot and Critchley polar plot were used to assess the accuracy and trending capability of the pulse co-oximeter. Data are presented as mean differences and 95% limits of agreement (LoA). RESULTS: In 101 data pairs analysed, the pulse co-oximeter slightly underestimated tHb (bias 0.06 g dL-1; LoA -1.0 to 1.2 g dL-1), SaO2 (bias 1.4%; LoA -2.0% to 4.8%), and CaO2 (bias 0.3 mL dL-1; LoA -2.1 to 2.7 mL dL-1). Zone A of the error grid encompassed 99% of data pairs for SpHb. Perfusion index (PI) ≥ 1% was recorded in 58/101 and PI < 1% in 43/101. The concordance rate for consecutive changes in SpHb and tHb with PI ≥ 1% and < 1% was 80% and 91% with four-quadrant plot, and 45.8% and 66.6% with Critchley polar plot. CONCLUSIONS: Pulse co-oximetry has acceptable accuracy for the values measured, even with low PI, whereas its trending ability requires further investigation in those horses with a higher [Hb] variation during anaesthesia.

The Emergence of RNA from the Heterogeneous Products of Prebiotic Nucleotide Synthesis.

Recent advances in prebiotic chemistry are beginning to outline plausible pathways for the synthesis of the canonical ribonucleotides and their assembly into oligoribonucleotides. However, these reaction pathways suggest that many noncanonical nucleotides are likely to have been generated alongside the standard ribonucleotides. Thus, the oligomerization of prebiotically synthesized nucleotides is likely to have led to a highly heterogeneous collection of oligonucleotides comprised of a wide range of types of nucleotides connected by a variety of backbone linkages. How then did relatively homogeneous RNA emerge from this primordial heterogeneity? Here we focus on nonenzymatic template-directed primer extension as a process that would have strongly enriched for homogeneous RNA over the course of multiple cycles of replication. We review the effects on copying the kinetics of nucleotides with altered nucleobase and sugar moieties, when they are present as activated monomers and when they are incorporated into primer and template oligonucleotides. We also discuss three variations in backbone connectivity, all of which are nonheritable and regenerate native RNA upon being copied. The kinetic superiority of RNA synthesis suggests that nonenzymatic copying served as a chemical selection mechanism that allowed relatively homogeneous RNA to emerge from a complex mixture of prebiotically synthesized nucleotides and oligonucleotides.

Thermally Driven Membrane Phase Transitions Enable Content Reshuffling in Primitive Cells.

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically relevant processes in a narrow range of nonfluctuating environmental conditions. Here we propose that membrane phase transitions, driven by environmental fluctuations, enabled the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures and the concomitant release of protocellular content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent nonfunctional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally driven tunable prebiotic process, which supports the release and reshuffling of oligonucleotides and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally driven disassembly/assembly cycle proposed herein would have plausibly supported protocellular content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early life forms.

DNA polymerase activity on synthetic N3'→P5' phosphoramidate DNA templates.

Genetic polymers that could plausibly govern life in the universe might inhabit a broad swath of chemical space. A subset of these genetic systems can exchange information with RNA and DNA and could therefore form the basis for model protocells in the laboratory. N3'→P5' phosphoramidate (NP) DNA is defined by a conservative linkage substitution and has shown promise as a protocellular genetic material, but much remains unknown about its functionality and fidelity due to limited enzymatic tools. Conveniently, we find widespread NP-DNA-dependent DNA polymerase activity among reverse transcriptases, an observation consistent with structural studies of the RNA-like conformation of NP-DNA duplexes. Here, we analyze the consequences of this unnatural template linkage on the kinetics and fidelity of DNA polymerization activity catalyzed by wild-type and variant reverse transcriptases. Template-associated deficits in kinetics and fidelity suggest that even highly conservative template modifications give rise to error-prone DNA polymerase activity. Enzymatic copying of NP-DNA sequences is nevertheless an important step toward the future study and engineering of this synthetic genetic polymer.

Inosine, but none of the 8-oxo-purines, is a plausible component of a primordial version of RNA.

The emergence of primordial RNA-based life would have required the abiotic synthesis of nucleotides, and their participation in nonenzymatic RNA replication. Although considerable progress has been made toward potentially prebiotic syntheses of the pyrimidine nucleotides (C and U) and their 2-thio variants, efficient routes to the canonical purine nucleotides (A and G) remain elusive. Reported syntheses are low yielding and generate a large number of undesired side products. Recently, a potentially prebiotic pathway to 8-oxo-adenosine and 8-oxo-inosine has been demonstrated, raising the question of the suitability of the 8-oxo-purines as substrates for prebiotic RNA replication. Here we show that the 8-oxo-purine nucleotides are poor substrates for nonenzymatic RNA primer extension, both as activated monomers and when present in the template strand; their presence at the end of a primer also strongly reduces the rate and fidelity of primer extension. To provide a proper comparison with 8-oxo-inosine, we also examined primer extension reactions with inosine, and found that inosine exhibits surprisingly rapid and accurate nonenzymatic RNA copying. We propose that inosine, which can be derived from adenosine by deamination, could have acted as a surrogate for G in the earliest stages of the emergence of life.

Comparison between dexmedetomidine and acepromazine in combination with methadone for premedication in brachycephalic dogs undergoing surgery for brachycephalic obstructive airway syndrome.

OBJECTIVE: To compare dexmedetomidine with acepromazine for premedication combined with methadone in dogs undergoing brachycephalic obstructive airway syndrome (BOAS) surgery. STUDY DESIGN: Randomized, blinded clinical study. ANIMALS: A group of 40 dogs weighing mean (± standard deviation) 10.5 ± 6 kg, aged 2.6 ± 1.9 years. METHODS: Dogs received either acepromazine 20 µg kg-1 (group A) or dexmedetomidine 2 µg kg-1 (group D) intramuscularly with methadone 0.3 mg kg-1. Anaesthesia was induced with propofol and maintained with sevoflurane. Sedation (0-18), induction (0-6) and recovery (0-5) qualities were scored. Propofol dose, hypotension incidence, mechanical ventilation requirement, extubation time, additional sedation, oxygen supplementation, regurgitation and emergency intubation following premedication or during recovery were recorded. Data were analysed using t tests, Mann-Whitney U or Chi-square tests. RESULTS: Group A dogs were less sedated [median (range): 1.5 (0-12)] than group D [5 (1-18)] (p = 0.021) and required more propofol [3.5 (1-7) versus 2.4 (1-8) mg kg-1; p = 0.018]. Induction scores [group A: 5 (4-5); group D 5 (3-5)] (p = 0.989), recovery scores [group A 5 (4-5); group D 5(3-5)](p = 0.738) and anaesthesia duration [group A:93 (50-170); group D 96 (54-263) minutes] (p = 0.758) were similar between groups. Time to extubation was longer in group A 12.5 (3-35) versus group D 5.5 (0-15) minutes; (p = 0.005). During recovery, two dogs required emergency intubation (p > 0.99) and five dogs required additional sedation (p > 0.99). Oxygen supplementation was required in 16 and 12 dogs in group A and D, respectively (p = 0.167); no dogs in group A and one dog in group D regurgitated (p = 0.311). CONCLUSIONS AND CLINICAL RELEVANCE: Dexmedetomidine 2 µg kg-1 produces more sedation but similar recovery quality to acepromazine 20 µg kg-1 combined with methadone in dogs undergoing BOAS surgery.

Assessment of pulse co-oximetry technology after in vivo adjustment in anaesthetized dogs.

OBJECTIVE: To compare values of haemoglobin concentration (SpHb), arterial haemoglobin saturation (SpO2) and calculated arterial oxygen content (SpOC), measured noninvasively with a pulse co-oximeter before and after in vivo adjustment (via calibration of the device using a measured haemoglobin concentration) with those measured invasively using a spectrophotometric-based blood gas analyser in anaesthetized dogs. STUDY DESIGN: Prospective observational clinical study. ANIMALS: A group of 39 adult dogs. METHODS: In all dogs after standard instrumentation, the dorsal metatarsal artery was catheterised for blood sampling, and a pulse co-oximeter probe was applied to the tongue for noninvasive measurements. Paired data for SpHb, SpO2 and SpOC from the pulse co-oximeter and haemoglobin arterial oxygen saturation (SaO2) and arterial oxygen content (CaO2) from the blood gas analyser were obtained before and after in vivo adjustment. Bland-Altman analysis for repeated measurements was used to evaluate the bias, precision and agreement between the pulse co-oximeter and the blood gas analyser. Data are presented as mean differences and 95% limits of agreement (LoA). RESULTS: A total of 39 data pairs were obtained before in vivo adjustment. The mean invasively measured haemoglobin-SpHb difference was -2.7 g dL-1 with LoA of -4.9 to -0.5 g dL-1. After in vivo adjustment, 104 data pairs were obtained. The mean invasively measured haemoglobin-SpHb difference was -0.2 g dL-1 with LoA of -1.1 to 0.6 g dL-1. The mean SaO2-SpO2 difference was 0.86% with LoA of -0.8% to 2.5% and that between CaO2-SpOC was 0.66 mL dL-1 with LoA of -2.59 to 3.91 mL dL-1. CONCLUSIONS: Before in vivo adjustment, pulse co-oximeter derived values overestimated the spectrophotometric-based blood gas analyser haemoglobin and CaO2 values. After in vivo adjustment, the accuracy, precision and LoA markedly improved. Therefore, in vivo adjustment is recommended when using this device to monitor SpHb in anaesthetised dogs.

Assembly of a Ribozyme Ligase from Short Oligomers by Nonenzymatic Ligation.

Our current understanding of the chemistry of the primordial genetic material is fragmentary at best. The chemical replication of oligonucleotides long enough to perform catalytic functions is particularly problematic because of the low efficiency of nonenzymatic template copying. Here we show that this problem can be circumvented by assembling a functional ribozyme by the templated ligation of short oligonucleotides. However, this approach creates a new problem because the splint oligonucleotides used to drive ribozyme assembly strongly inhibit the resulting ribozyme. We explored three approaches to the design of splint oligonucleotides that enable efficient ligation but which allow the assembled ribozyme to remain active. DNA splints, splints with G:U wobble pairs, and splints with G to I (Inosine) substitutions all allowed for the efficient assembly of an active ribozyme ligase. Our work demonstrates the possibility of a transition from nonenzymatic ligation to enzymatic ligation and reveals the importance of avoiding ribozyme inhibition by complementary oligonucleotides.

A Model for the Emergence of RNA from a Prebiotically Plausible Mixture of Ribonucleotides, Arabinonucleotides, and 2'-Deoxynucleotides.

The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2'-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2'-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2'-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2'-deoxy- or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

Template-Directed Copying of RNA by Non-enzymatic Ligation.

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. The rate of ligation can be greatly enhanced by employing a 3'-amino group at the 3'-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution.

Nonenzymatic Template-Directed Synthesis of Mixed-Sequence 3'-NP-DNA up to 25 Nucleotides Long Inside Model Protocells.

Efficiently copying mixed-sequence oligonucleotide templates nonenzymatically is a long-standing problem both with respect to the origin of life, and with regard to bottom up efforts to synthesize artificial living systems. Here we report an efficient and sequence-general nonenzymatic process in which RNA templates direct the synthesis of a complementary strand composed of N3'→P5' phosphoramidate DNA (3'-NP-DNA) using 3'-amino-2',3'-dideoxyribonucleotides activated with 2-aminoimidazole. Using only the four canonical nucleobases (A, G, C, and T) of modern DNA, we demonstrate the chemical copying of a variety of mixed-sequence RNA templates, both in solution and within model protocells, into complementary 3'-NP-DNA strands. Templates up to 25 nucleotides long were chemically transcribed with an average stepwise yield of 96-97%. The nonenzymatic template-directed generation of primer extension products long enough to encode active ribozymes and/or aptamers inside model protocells suggests possible routes to the synthesis of evolving cellular systems.

Prebiotically Plausible "Patching" of RNA Backbone Cleavage through a 3'-5' Pyrophosphate Linkage.

Achieving multiple cycles of RNA replication within a model protocell would be a critical step toward demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an "RNA world" must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an end point for RNA replication. Instead, 3'-phosphate-terminated RNA strands can participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. The presence of free magnesium cations results in cleavage of the pyrophosphate bond within minutes. However, we found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. We show that, under these conditions, pyrophosphate-linked RNA can act as a template for the polymerization of ribonucleotides into canonical 3'-5' phosphodiester-linked RNA. We suggest that primer extension of 3'-phosphate-terminated RNA followed by template-directed copying represents a plausible nonenzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

Copying of Mixed-Sequence RNA Templates inside Model Protocells.

The chemical replication of RNA inside fatty acid vesicles is a plausible step in the emergence of cellular life. On the primitive Earth, simple protocells with the ability to import nucleotides and short oligomers from their environment could potentially have replicated and retained larger genomic RNA oligonucleotides within a spatially defined compartment. We have previously shown that short 5'-phosphoroimidazolide-activated "helper" RNA oligomers enable the nonenzymatic copying of mixed-sequence templates in solution, using 5'-phosphoroimidazolide-activated mononucleotides. Here, we report that citrate-chelated Mg2+, a catalyst of nonenzymatic primer extension, enhances fatty acid membrane permeability to such short RNA oligomers up to the size of tetramers, without disrupting vesicle membranes. In addition, selective permeability of short, but not long, oligomers can be further enhanced by elevating the temperature. The ability to increase the permeability of fatty acid membranes to short oligonucleotides allows for the nonenzymatic copying of RNA templates containing all four nucleotides inside vesicles, bringing us one step closer to the goal of building a protocell capable of Darwinian evolution.

O4 -Alkylated-2-Deoxyuridine Repair by O6 -Alkylguanine DNA Alkyltransferase is Augmented by a C5-Fluorine Modification.

Oligonucleotides containing various adducts, including ethyl, benzyl, 4-hydroxybutyl and 7-hydroxyheptyl groups, at the O4 atom of 5-fluoro-O4 -alkyl-2'-deoxyuridine were prepared by solid-phase synthesis. UV thermal denaturation studies demonstrated that these modifications destabilised the duplex by approximately 10 °C, relative to the control containing 5-fluoro-2'-deoxyuridine. Circular dichroism spectroscopy revealed that these modified duplexes all adopted a B-form DNA structure. O6 -Alkylguanine DNA alkyltransferase (AGT) from humans (hAGT) was most efficient at repair of the 5-fluoro-O4 -benzyl-2'-deoxyuridine adduct, whereas the thymidine analogue was refractory to repair. The Escherichia coli AGT variant (OGT) was also efficient at removing O4 -ethyl and benzyl adducts of 5-fluoro-2-deoxyuridine. Computational assessment of N1-methyl analogues of the O4 -alkylated nucleobases revealed that the C5-fluorine modification had an influence on reducing the electron density of the O4 -Cα bond, relative to thymine (C5-methyl) and uracil (C5-hydrogen). These results reveal the positive influence of the C5-fluorine atom on the repair of larger O4 -alkyl adducts to expand knowledge of the range of substrates able to be repaired by AGT.

Covalent capture of OGT's active site using engineered human-E. coli chimera and intrastrand DNA cross-links.

O 6-Alkylguanine DNA alkyltransferases (AGTs) are proteins found in most organisms whose role is to remove alkylation damage from the O6- and O4-positions of 2'-deoxyguanosine (dG) and thymidine (dT), respectively. Variations in active site residues between AGTs from different organisms leads to differences in repair proficiency: The human variant (hAGT) has a proclivity for removal of alkyl groups at the O6-position of guanine and the E. coli OGT protein has activity towards the O4-position of thymine. A chimeric protein (hOGT) that our laboratory has engineered with twenty of the active site residues mutated in hAGT to those found in OGT, exhibited activity towards a broader range of substrates relative to native OGT. Among the substrates that the hOGT protein was found to act upon was interstrand cross-linked DNA connected by an alkylene linkage at the O6-position of dG to the complementary strand. In the present study the activity of hOGT towards DNA containing alkylene intrastrand cross-links (IaCL) at the O6- and O4-positions respectively of dG and dT, which lack a phosphodiester linkage between the connected residues, was evaluated. The hOGT protein exhibited proficiency at removal of an alkylene linkage at the O6-atom of dG but the O4-position of dT was refractory to protein activity. The activity of the chimeric hOGT protein towards these IaCLs to prepare well defined DNA-protein cross-linked conjugates will enable mechanistic and high resolution structural studies to address the differences observed in the repair adeptness of O4-alkylated dT by the OGT protein relative to other AGT variants.

A Fluorescent G-Quadruplex Sensor for Chemical RNA Copying.

Non-enzymatic RNA replication may have been one of the processes involved in the appearance of life on Earth. Attempts to recreate this process in a laboratory setting have not been successful thus far, highlighting a critical need for finding prebiotic conditions that increase the rate and the yield. Now a highly parallel assay for template directed RNA synthesis is presented that relies on the intrinsic fluorescence of a 2-aminopurine modified G-quadruplex. The application of the assay to examine the combined influence of multiple variables including pH, divalent metal concentrations and ribonucleotide concentrations on the copying of RNA sequences is demonstrated. The assay enables a direct survey of physical and chemical conditions, potentially prebiotic, which could enable the chemical replication of RNA.