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 Oligomerization of Ribonucleotides: Towards in vitro Selection Experiments

Inspired by the polymerase chain reaction, orthogonal primer–template pairs have been applied in template‐controlled oligomerization experiments with Orgel's imidazole‐activated ribonucleotide‐5′‐phosphates. Variation of the linker length allowed us to monitor the extension of both primers simultaneously on a DNA sequencer. Sets of hexapyrimidine primers were found that are capable of inducing the reciprocal synthesis of each other's binding site. Considerable cross‐inhibition by different monomers was observed. However, this effect is a function of primer sequence and can disappear in favorable cases. With random sequences introduced into the templates, selection experiments are within reach. First results are reported below.     

Activated ribonucleotides undergo a sugar pucker switch upon binding to a single-stranded RNA template

Template-directed polymerization of chemically activated ribonucleotide monomers, such as nucleotide 5'-phosphorimidazolides, has been studied as a model for nonenzymatic RNA replication during the origin of life. Kinetic studies of the polymerization of various nucleotide monomers on oligonucleotide templates have suggested that the A-form (C3'-endo sugar pucker) conformation is optimal for both monomers and templates for efficient copying. However, RNA monomers are predominantly in the C2'-endo conformation when free in solution, except for cytidine, which is approximately equally distributed between the C2'-endo and C3'-endo conformations. We hypothesized that ribonucleotides undergo a switch in sugar pucker upon binding to an A-type template and that this conformational switch allows or enhances subsequent polymerization. We used transferred nuclear Overhauser effect spectroscopy (TrNOESY), which can be used for specific detection of the bound conformation of small-molecule ligands with relatively weak affinity to receptors, to study the interactions between nucleotide 5'-phosphorimidazolides and single-stranded oligonucleotide templates. We found that the sugar pucker of activated ribonucleotides switches from C2'-endo in the free state to C3'-endo upon binding to an RNA template. This switch occurs only on RNA and not on DNA templates. Furthermore, activated 2'-deoxyribonucleotides maintain a C2'-endo sugar pucker in both the free and template-bound states. Our results provide a structural explanation for the observations that activated ribonucleotides are superior to activated deoxyribonucleotides and that RNA templates are superior to DNA templates in template-directed nonenzymatic primer-extension reactions.     

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.     

Application of Single—labelled Probe—primer in PCR Amplification to the Detection of Hepatitis B Virus DNA

A new method based on the incorporation of a single-lablled probe-primer into polymerase chain reaction(PCR) for the detection of PCR-amplified DNA in a closed system is reported.The probeprimerc consists of a specific probe sequence on the 5‘‘‘‘‘‘‘‘-end and a primer sequence on the 3‘‘‘‘‘‘‘‘-end.A flurophore is located at the 5‘‘‘‘‘‘‘‘end.The primeR-quencher is an oligonucleotide,which is complementary to the probe sequence of probe-primer and labelled with a quencher at the 3‘‘‘‘‘‘‘‘-end.In the duplex formed by probe-primer and primer-quencher.the fluorophore and quencher are kept in close proximity to each other.Therefore the fluorescence is quenched.During PCR amplificatio,the specific probe sequence of probeprimer binds to its complement within the same strand of DNA,and is cleaved by Taq DNA polymerase,resulting in the restoration of fluorescence.This system has the same energy transfer mechanism as molecular beacons,and a good quenching effciency can be ensured.Following optimization of PCR conditions,this method was used to detect hepatitis b virus(HBV) dna in patient sera.This technology eliminates the risk of carry-over contamination,simplifies the amplification assay and opens up new possibilities for the real-time detection of the amplified DNA.     

Enzymatic Fabrication of High‐Density RNA Arrays

A powerful new strategy for the fabrication of high‐density RNA arrays is described. A high‐density DNA array is fabricated by standard photolithographic methods, the surface‐bound DNA molecules are enzymatically copied into their RNA complements from a surface‐bound RNA primer, and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. The strategy is compatible with 2′‐fluoro‐modified (2′F) ribonucleoside triphosphates (rNTPs), which may be included in the polymerase extension reaction to impart nuclease resistance and other desirable characteristics to the synthesized RNAs. The use and fidelity of the arrays are explored with DNA hybridization, DNAzyme cleavage, and nuclease digestion experiments.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

?29 DNA polymerase (?29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ?29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ?29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

ϕ29 DNA polymerase (ϕ29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ϕ29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ϕ29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

Pyranosyl-RNA: chiroselective self-assembly of base sequences by ligative oligomerization of tetra nucleotide-2′,3′-cyclophosphates (with a commentary concerning the origin of biomolecular homochirality)

Background: Why did Nature choose furanosyl-RNA and not pyranosyl-RNA as her molecular genetic system? An experimental approach to this problem is the systematic comparison of the two isomeric oligonucleotide systems with respect to the chemical properties that are fundamental to the biological role of RNA, such as base pairing and nonenzymic replication. Pyranosyl-RNA has been found to be not only a stronger, but also a more selective pairing system than natural RNA; both form hairpin structures with comparable ease. Base sequences of pyranosyl-RNA can be copied by template-controlled replicatioe ligation of short activated oligomers (e.g. tetramer-2′,3′-cyclophosphates) under mild and potentially natural conditions. The copying proceeds with high regio-selectivity as well as chiroselectivity: homochiral template sequences mediate the formation of the correct (4′→2′)-phosphodiester junction between homochiral tetramer units provided they have the same sense of chirality as the template. How could homochiral template sequences assemble themselves in the first place?Results: Higher oligomers of pyranosyl-RNA can self-assemble in dilute solutions under mild conditions by ligative oligomerization of tetramer-2′,3′-cyclophosphates containing hemi self-complementary base sequences. The only side reaction that effectively competes with ligation is hydrolytic deactivation of 2′,3′-cyclophosphate end groups. The ligation reaction is highly chiroselective; it is slower by at least two orders of magnitude when one of the (d)-ribopyranosyl units of a homochiral (d)-tetramer-2′,3′-cyclophosphate is replaced by a corresponding (l)-unit, except when the (l)-unit is at the 4′ end of the tetramer and carries a purine, when the oligomerization rate can be ∼ 10% of that shown for a homochiral isomer. The oligomerization of homochiral tetramers is not, or only weakly, inhibited by the presence of the non-oligomerizing diastereomers.Conclusions: Available data on the chiroselective self-directed oligomerization of tetramer-2′,3′-cyclophosphates allow us to extrapolate that sets of tetramers with different but mutually fitting base sequences can be expected to co-oligomerize stochastically and generate sequence libraries consisting of predominantly homochiral (d)- and (l)-oligomers, starting from the racemic mixture of tetramers containing all possible diastereomers. Such a capability of an oligonucleotide system deserves special attention in the context of the problem of the origin of biomolecular homochirality: breaking molecular mirror symmetry by de-racemization is an intrinsic property of such a system whenever the constitutional complexity of the products of co-oligomerization exceeds a critical level.     

Squaramate‐Modified Nucleotides and DNA for Specific Cross‐Linking with Lysine‐Containing Peptides and Proteins

Squaramate‐linked 2′‐deoxycytidine 5′‐O‐triphosphate was synthesized and found to be good substrate for KOD XL DNA polymerase in primer extension or PCR synthesis of modified DNA. The resulting squaramate‐linked DNA reacts with primary amines to form a stable diamide linkage. This reaction was used for bioconjugations of DNA with Cy5 and Lys‐containing peptides. Squaramate‐linked DNA formed covalent cross‐links with histone proteins. This reactive nucleotide has potential for other bioconjugations of nucleic acids with amines, peptides or proteins without need of any external reagent.     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

ULTRAVIOLET INACTIVATION OF THE MIDI VARIANT OF QP RNA: CHARACTERIZATION OF TEMPLATE ACTIVITY

Abstract— MDV-1 RNA is a 218 nucleotide variant of bacteriophage Qβ RNA. Qβ replicase catalyzes the formation of a strand complementary to a single-stranded (SS) MDV-I template. Upon phenol extraction, the template and complementary strands become double-stranded (DS). Polyacrylamide gel electrophoresis of the products of this reaction revealed SS RNA, DS RNA, and discrete intermediate bands. UV irradiation of the template caused a decrease in DS RNA production which followed single-hit kinetics with a quantum yield of 1.6 × 10^--³. Concomitant with this diminished DS RNA production were increases in SS RNA and intermediate sized RNA. The latter was shown to consist of a full sized SS template annealed to a partially completed nascent strand. Upon electrophoresis, these partially completed duplexes migrated in the same positions as those found in the analysis of unirradiated template, suggesting that this RNA contains replication obstruction areas in which UV lesions cause an increase in replication inhibition.     

Substrate 2'-hydroxyl groups required for ribozyme-catalyzed polymerization

A polymerase ribozyme has been generated that uses nucleoside triphosphates to elongate an RNA primer by the successive addition of nucleotides complementary to an RNA template. Its polymerization is accurate, with an average error rate less than 3%, and it is general in terms of the sequence and the length of the primer and template RNAs. To begin to understand how the substrate contacts contribute to this accurate and general activity, we investigated which primer and template 2'-hydroxyl groups are involved in substrate recognition. We identified eight positions where 2'-deoxy substitutions can influence polymerization kinetics. All eight are within five nucleotides of the primer 3' terminus. Some, but not all, of the 2'-deoxy effects appear to be sequence dependent. These results begin to build a picture of how the polymerase ribozyme recognizes its substrates.     

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.     

Spontaneous Aminoacylation of a RNA Sequence Containing a 3′‐Terminal 2′‐Thioadenosine

We report the synthesis of a modified 8mer RNA sequence, (C‐C‐C‐C‐A‐C‐C‐(2′‐thio)A)‐RNA 5′‐(dihydrogen phosphate) ( 9 ) containing a 3′‐terminal 2′‐thioadenosine (Schemes 2 and 3), and its spontaneous and site‐specific aminoacylation with the weakly activated amino acid thioester H Phe SPh ( 12 ). This reaction, designed in analogy to the ‘native chemical ligation’ of oligopeptides, occurs efficiently in buffered aqueous solutions and under a wide range of conditions (Table). At pH values between 5.0 and 7.4, two products, the 3′‐O‐monoacylated and the 3′‐O,2′‐S‐diacylated RNA sequences 10 and 11 are formed fast and quantitatively (Scheme 4). At pH 7.4 and 37°, the 3′‐O‐monoacylated product 10 is formed as major product in situ by selective hydrolysis of the O,S‐diacylated precursor 11 . Additionally, the preparation and isolation of the relevant 3′‐O‐monoacylated product 10 was optimized at pH 5. The here presented concept could be employed for a straightforward aminoacylation of analogously modified tRNAs.     

Intramolecular Participation of Amino Groups in the Cleavage and Isomerization of Ribonucleoside 3′‐Phosphodiesters: The Role in Stabilization of the Phosphorane Intermediate

A dinucleoside‐3′,5′‐phosphodiester model, 5′‐amino‐4′‐aminomethyl‐5′‐deoxyuridylyl‐3′,5′‐thymidine, incorporating two aminomethyl functions in the 4′‐position of the 3′‐linked nucleoside has been prepared and its hydrolytic reactions studied over a wide pH range. The amino functions were found to accelerate the cleavage and isomerization of the phosphodiester linkage in both protonated and neutral form. When present in protonated form, the cleavage of the 3′,5′‐phosphodiester linkage and its isomerization to a 2′,5′‐linkage are pH‐independent and 50–80 times as fast as the corresponding reactions of uridylyl‐3′,5′‐uridine (3′,5′‐UpU). The cleavage of the resulting 2′,5′‐isomer is also accelerated, albeit less than with the 3′,5′‐isomer, whereas isomerization back to the 3′,5′‐diester is not enhanced. When the amino groups are deprotonated, the cleavage reactions of both isomers are again pH‐independent and up to 1000‐fold faster than the pH‐independent cleavage of UpU. Interestingly, the 2′‐ to 3′‐isomerization is now much faster than its reverse reaction. The mechanisms of these reactions are discussed. The rate accelerations are largely accounted for by electrostatic and hydrogen‐bonding interactions of the protonated amino groups with the phosphorane intermediate.     

Covalent Chemical 5′‐Functionalization of RNA with Diazo Reagents

Functionalization of RNA at the 5′‐terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5′‐nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5′‐phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5′‐end of RNA.