Clemens Richert

Efficient enzyme-free copying of all four nucleobases templated by immobilized RNA

The transition from inanimate materials to the earliest forms of life must have involved multiplication of a catalytically active polymer that is able to replicate. The semiconservative replication that is characteristic of genetic information transfer requires strands that contain more than one type of nucleobase. Short strands of RNA can act as catalysts, but attempts to induce efficient self-copying of mixed sequences (containing four different nucleobases) have been unsuccessful with ribonucleotides. Here we show that inhibition by spent monomers, formed by the hydrolysis of the activated nucleotides, is the cause for incomplete extension of growing daughter strands on RNA templates. Immobilization of strands and periodic displacement of the solution containing the activated monomers overcome this inhibition. Any of the four nucleobases (A/C/G/U) is successfully copied in the absence of enzymes. We conclude therefore that in a prebiotic world, oligoribonucleotides may have formed and undergone self-copying on surfaces.

Modifications in Small Interfering RNA That Separate Immunostimulation from RNA Interference

Synthetic small interfering RNA (siRNA) can suppress the expression of endogenous mRNA through RNA interference. It has been reported that siRNA can induce type I IFN production from plasmacytoid dendritic cells, leading to off-target effects. To separate immunostimulation from the desired gene-specific inhibitory activity, we designed RNA strands with chemical modifications at strategic positions of the ribose or nucleobase residues. Substitution of uridine residues by 2′-deoxyuridine or thymidine residues was found to decrease type I IFN production upon in vitro stimulation of human PBMC. Thymidine residues in both strands of a siRNA duplex further decreased immunostimulation. Fortunately, the thymidine residues did not affect gene-silencing activity. In contrast, 2′-O-methyl groups at adenosine and uridine residues reduced both IFN-α secretion and gene-silencing activity. Oligoribonucleotides with 2′-O-methyladenosine residues actively inhibited IFN-α secretion induced by other immunostimulatory RNAs, an effect not observed for strands with 2′-deoxynucleosides. Furthermore, neither 5-methylcytidine nor 7-deazaguanosine residues in the stimulatory strands affected IFN-α secretion, suggesting that recognition does not involve sites in the major groove of duplex regions. The activity data, together with structure prediction and exploratory UV-melting analyses, suggest that immunostimulatory sequences adopt folded structures. The results show that immunostimulation can be suppressed by suitable chemical modifications without losing siRNA potency by introducing seemingly minor structural changes.

Rapid genotyping by MALDI-monitored nuclease selection from probe libraries.

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5′-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (≥125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).

Branched DNA That Forms a Solid at 95 °C

Control over the structure of materials may be achieved by using predictable interactions, such as base pairing. Base pairing between DNA strands is emerging as one of the most versatile design principles of nanoconstruction. A range of hybridization and folding motifs of linear and circular DNA have been reported. The flexibility of the design has been further expanded by linking oligonucleotides to synthetic branching elements or “cores”. 5] The resulting construct can have properties not found in natural DNA. This includes DNA-coated gold nanoparticles that assemble into three-dimensional aggregates, the melting transitions of which are exceptionally sharp. Nanoparticle size and linker structure affect the association behavior, and crystallization may be induced in favorable cases. For DNA hybrids with organic cores, the effect of linking the DNA to a branching element can be more dramatic still. Four-arm hybrid 1 (Scheme 1) with its tetrahedral core was recently shown to assemble into a macroscopic material, even though its oligonucleotide arms are just dimers. The assembly process is sequence specific, as demonstrated by mismatch controls, but the UV-melting transitions are broad, not sharp as in the case of gold nanoparticles. Shortly after the publication of the unusually stable assemblies of 1, the first designed DNA crystals were reported. The fact that the association of the rigid triangle motifs that serve as rigid “cores” in these crystals is also driven by no more than dimer “sticky ends” again suggests that the rules for 3D construction of periodic assemblies are quite different from those of linear DNA.

Quantitative MALDI-TOF MS of oligonucleotides and a nuclease assay

Abstract DNA oligomers 8 and 12 nucleotides long have been detected quantitatively by MALDI-TOF mass spectrometry using longer oligonucleotides as internal standards. Employing this method, the kinetics of the nuclease degradation of the octamer were analyzed. Reactions performed in the presence of the ssDNA-binding peptide KWK yielded a footprint based on fragments from both the 3′- and the 5′-terminus of the oligonucleotide.

Two Base Pair Duplexes Suffice to Build a Novel Material

Tetrahedral DNA hybrids with tetrakis(p‐hydroxyphenyl)methane cores hybridize in a sequence‐specific fashion at much higher temperatures than isolated linear duplexes. Dinucleotide DNA arms suffice to induce the formation of a solid at room temperature; this demonstrates the strength of multivalent binding. The graphic shows a view of a modeled assembly.

Chemical Primer Extension in Seconds

Chemical primer extension (CPE) generates nucleic acid strands elongated by the nucleotides encoded in a template strand in the absence of enzymes. Enzymatic primer extension is a fundamental reaction that underlies both replication and transcription. It is also pivotal for biotechnology, as PCR and dideoxy sequencing are based on primer extension. Furthermore, the formation and template-directed extension of primers is probably a key process in the prebiotic phase of the evolution of life. CPE can produce sequence information with unlabeled or fluorophore-labeled mononucleotides. Compared to polymerase-catalyzed primer extension, CPE is inexpensive as it avoids the cost of enzymes and triphosphate substrates, which makes it attractive for genotyping and sequencing applications. A reduction in cost is critical to meet the

Redesigning nucleic acids

1000 genome challenge. The chemical replication or copying reactions underlying CPE have been studied for decades in the context of prebiotic evolution. A drawback of known forms of these reactions is that they are less efficient and much slower than their enzymatically catalyzed counterparts. Polymerase-catalyzed primer extension leads to replication of entire bacterial genomes within minutes, but chemical copying assays are run on the timescale of days to weeks. Attempts have been made to overcome the slow nature of primer generation and CPE steps. Oxyazabenzotriazolides were identified as reactive monomers that reduce the half-life time (t1/2) of CPE involving amino-terminal DNA or RNA by more than an order of magnitude, but all subsequent attempts to accelerate these reactions further appeared to hit a “solid wall” of unreactivity or lead to an unacceptable level of lability among the monomers. Control experiments meant to test the tolerance of the assay for pyridine (whose presence was needed to allow for direct addition of active esters of monomers generated in this solvent) led to an unexpected result. At high millimolar concentrations, pyridine massively accelerates spontaneous replication steps by what appears to be an organocatalytic process. The range of reactions for which “organocatalysis” has been established has increased substantially. Although the phenomenon has been known for a long time, the focus of contemporary research has been on enantioselective reactions catalyzed by substoichiometric amounts of chiral organic molecules. Reactions as different mechanistically as aldol additions and transfer hydrogenation benefit from the presence of organocatalysts. However, transphosphorylation reactions are not among the synthetic transformations frequently catalyzed by organocatalysts. Instead, activation by metal ions, such as the magnesium ions found in the active site of polymerases or minerals, is more common. On the other hand, solid-phase DNA synthesis became rapid (and a successful commercial process) when phosphoramidites were combined with tetrazole as organocatalyst. This catalyst is usually referred to as an “activator”, as it is used above stoichiometric concentration. Our study on CPE employed oxyazabenzotriazolides of deoxynucleotides (1a, 1c, 1g, 1t) and 3’-amino-terminal primers (Scheme 1) to generate phosphoramidate linkages that are isoelectronic to phosphodiesters. Some phosphoramidates are accepted by polymerases. Oxyazabenzotriazolides produce phosphodiesters in RNA-based reactions. ,21] Earlier attempts to employ known catalysts for ligation in this system, including proflavine, had been essentially unsuccessful. For our screens, we transferred the assays involving 1 a–t to magnetic beads (Scheme 2). Excess monomers and buffer

Templating efficiency of naked DNA

A research program has applied the tools of synthetic organic chemistry to systematically modify the structure of DNA and RNA oligonucleotides to learn more about the chemical principles underlying their ability to store and transmit genetic information. Oligonucleotides (as opposed to nucleosides) have long been overlooked by synthetic organic chemists as targets for structural modification. Synthetic chemistry has now yielded oligonucleotides with 12 replicatable letters, modified backbones, and new insight into why Nature chose the oligonucleotide structures that she did.

A CONVENIENT SYNTHESIS OF 5'-AMINO-5'-DEOXYTHYMIDINE AND PREPARATION OF PEPTIDE-DNA HYBRIDS

Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with ≥93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows ≥72% of the correct extension product in all cases, and ≥90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.