The nitroxide-containing nucleoside Çm is reported as the first rigid spin label for paramagnetic modification of RNA by solid-phase synthesis. The spin label is well accommodated in several RNA secondary structures as judged by its minor effect on the thermodynamic stability of hairpin and duplex RNA. Electron paramagnetic resonance (EPR) spectroscopic characterization of mono-, bi-, and trimolecular RNA structures shows that Çm will be applicable for advanced EPR studies to elucidate structural and dynamic aspects of folded RNA.
The ability of RNA to interconvert between multiple conformational states is essential for the diversity of biological functions that have been discovered in the recent past. [1] For example, the correct operation of regulatory RNA elements, such as riboswitches, is based on the precise interplay of alternative RNA conformations.[2] Studying the molecular mechanisms of RNA function entails probing RNA-folding intermediates on the energy landscape. EPR spectroscopy, in particular, has been increasingly applied to obtain structural information on nucleic acids, including local conformational changes in RNA [3] and the identification of metal-ion binding sites.[4] Pulsed EPR techniques (PELDOR/ DEER) have been used to determine distances between paramagnetic centers in specifically modified RNA. [5] PELDOR should therefore be suitable for the detection of alternative RNA conformations that involve distinct changes in base-pairing patterns.The accessibility of spin-labeled RNA still poses the major challenge for the widespread applicability of powerful EPR techniques. Nitroxide radicals are the most commonly used type of paramagnetic labels for nucleic acids. Several methods have been reported for attaching nitroxide groups at internal positions at the ribose, the phosphate backbone, or at nucleobases, often by means of multiatom linkers that provide several unwanted degrees of rotational freedom.[6] Rigid nitroxide spin labels conjugated to the nucleobase or to nucleobase analogues have been reported for DNA.[7] Our RNA spin-labeling approach addresses the direct attachment of nitroxide labels onto RNA nucleobases, such that conformational changes can be directly detected by PELDOR (i.e., by the change in distance between two labeled nucleotides). The nucleobase spin labels used in this study are also designed to preserve the Watson-Crick base-pairing capability of labeled nucleotides and not to interfere with alternative base-pairing patterns in different RNA conformations.Here, we describe the installation of nitroxide spin labels on exocyclic amino groups of the RNA nucleobases guanine, adenine, and cytosine (Figure 1) with unprecedented efficiency, and we report on the evaluation of RNA secondary structures by pulsed double electron resonance spectroscopy.The convertible nucleosides [8] O 4 -(4-chlorophenyl)uridine, O 6 -(4-chlorophenyl)inosine, and 2-fluoroinosine were incorporated into RNA by solid-phase synthesis using commercially available, modified 2'-O-tert-butyldimethylsilylprotected building blocks 1-3 in combination with standard RNA 2'-O-triisopropylsilyloxymethyl-protected nucleoside phosphoramidites. In a postsynthetic modification step, the 4-chlorophenyl or fluoride leaving groups were displaced by
The emergence of functional cooperation between the three main classes of biomolecules - nucleic acids, peptides and lipids - defines life at the molecular level. However, how such mutually interdependent molecular systems emerged from prebiotic chemistry remains a mystery. A key hypothesis, formulated by Crick, Orgel and Woese over 40 year ago, posits that early life must have been simpler. Specifically, it proposed that an early primordial biology lacked proteins and DNA but instead relied on RNA as the key biopolymer responsible not just for genetic information storage and propagation, but also for catalysis, i.e. metabolism. Indeed, there is compelling evidence for such an 'RNA world', notably in the structure of the ribosome as a likely molecular fossil from that time. Nevertheless, one might justifiably ask whether RNA alone would be up to the task. From a purely chemical perspective, RNA is a molecule of rather uniform composition with all four bases comprising organic heterocycles of similar size and comparable polarity and pK a values. Thus, RNA molecules cover a much narrower range of steric, electronic and physicochemical properties than, e.g. the 20 amino acid side-chains of proteins. Herein we will examine the functional potential of RNA (and other nucleic acids) with respect to self-replication, catalysis and assembly into simple protocellular entities.
Central to the “RNA world” hypothesis of the origin of life is the emergence of an RNA catalyst capable of RNA replication. However, possible replicase ribozymes are quite complex and were likely predated by simpler non-enzymatic replication reactions. The templated polymerisation of phosphorimidazolide (Imp) activated ribonucleotides currently appears as the most tractable route to both generate and replicate short RNA oligomer pools from which a replicase could emerge. Herein we demonstrate the rapid assembly of complex ribozymes from such Imp-activated RNA fragment pools. Specifically, we show assembly of a newly selected minimal RNA polymerase ribozyme variant (150 nt) by RNA templated ligation of 5’-2-methylimidazole-activated RNA oligomers <30 nucleotides long. Our results provide support for the possibility that complex RNA structures could have emerged from pools of activated RNA oligomers and outlines a path for the transition from non-enzymatic/chemical to enzymatic RNA replication.
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