The intricate world of riboswitches.

Riboswitches are segments of the 5'-untranslated region of certain bacterial mRNAs that upon recognition of specific ligands modify the expression of a protein(s) encoded in the message. These proteins are responsible for the biosynthesis or transport of ligands, which are typically organic molecules but could also be metal ions. Riboswitch-mediated control of gene expression might be thermodynamic or kinetic, depending on the rate of transcription elongation by RNA polymerase and the structures adopted by the riboswitch RNA.

Mimicking the first step of RNA splicing: an artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5'-exon analogue.

The 7S11 deoxyribozyme synthesizes 2',5'-branched RNA by mediating the nucleophilic attack of an internal 2'-hydroxyl group of one RNA substrate into the 5'-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5'-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5'-diphosphate or 5'-monophosphate, the same 2',5'-branched product is formed as when pyrophosphate is the leaving group, but with an approximately 50- or approximately 1000-fold lower rate (Brønsted beta(LG) = -0.

General deoxyribozyme-catalyzed synthesis of native 3'-5' RNA linkages.

An elusive goal for nucleic acid enzymology has been deoxyribozymes that ligate RNA rapidly, sequence-generally, with formation of native 3'-5' linkages, and in preparatively useful yield. Using in vitro selection, we have identified Mg2+- and Zn2+-dependent deoxyribozymes that simultaneously fulfill all four of these criteria. The new deoxyribozymes operate under practical incubation conditions and have modest RNA substrate sequence requirements, specifically D downward arrowRA for 9DB1 and A downward arrowR for 7DE5 (D = A, G, or U; R = A or G).

A deoxyribozyme that forms a three-helix-junction complex with its RNA substrates and has general RNA branch-forming activity.

We recently used in vitro selection to identify 7S11, a deoxyribozyme that synthesizes 2',5'-branched RNA. The 7S11 DNA enzyme mediates the nucleophilic attack of an adenosine 2'-hydroxyl group at a 5'-triphosphate, forming 2',5'-branched RNA in a reaction that resembles the first step of in vivo RNA splicing. Here, we describe 7S11 characterization experiments that have two important implications for nucleic acid chemistry and biochemistry.

Rational modification of a selection strategy leads to deoxyribozymes that create native 3'-5' RNA linkages.

We previously used in vitro selection to identify several classes of deoxyribozymes that mediate RNA ligation by attack of a hydroxyl group at a 5'-triphosphate. In these reactions, the nucleophilic hydroxyl group is located at an internal 2'-position of an RNA substrate, leading to 2',5'-branched RNA. To obtain deoxyribozymes that instead create linear 3'-5'-linked (native) RNA, here we strategically modified the selection approach by embedding the nascent ligation junction within an RNA:DNA duplex region.

A DNA enzyme that mimics the first step of RNA splicing.

We have discovered an artificial DNA enzyme that mimics the first step of RNA splicing. In vitro selection was used to identify DNA enzymes that ligate RNA. One of the new DNA enzymes carries out splicing-related catalysis by specifically recognizing an unpaired internal adenosine and facilitating attack of its 2'-hydroxyl onto a 5'-triphosphate.

Deoxyribozymes with 2'-5' RNA ligase activity.

In vitro selection was used to identify deoxyribozymes that ligate two RNA substrates. In the ligation reaction, a 2'-5' RNA phosphodiester linkage is created from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The new Mg(2+)-dependent deoxyribozymes provide 50-60% yield of ligated RNA in overnight incubations at pH 7.