Ribozymes: RNA tools and therapeutic applications

• Ribozymes are enzymes that can modify RNA and DNA sequences
• Ribozymes can be used as RNA scissors, riboswitches, genome editing reagents, drug targets, and RNA drugs for therapies
• Ribozymes can be synthesized in a laboratory

Hammerheads-RNA Scissors

• Hammerheads are RNA scissors that can remove unwanted transcripts

Group I Introns Therapeutic Applications

• Ribozyme-based gene therapies are developed for incurable diseases, from genetic disorders to viral infections to cancers
• Ribozymes downregulate or repair pathogenic genes via RNA cleavage or reprograming facilitated by trans-splicing ribozymes
• Trans-splicing ribozymes edit target RNAs through simultaneous destruction and repair, inducing therapeutic gene activity in specified cells
• Trans-splicing ribozymes can reprogram specific RNAs in organisms via 3'-tagging of reporter RNAs, and are used in molecular imaging technologies

Group II Introns

• Many labs work on designing targetrons, which use mobile introns for genetic engineering
• CRISPR/Cas9 is becoming the dominant genome editing tool for Eukaryotic systems
• Group II introns undergo splicing and reverse splicing
• Exon binding sequence is abbreviated EBS
• Intron binding sequence is IBS

Group II Intron Retrohoming

• Group II intron retrohoming occurs in the first step when the reverse transcriptase (RT) binds to its cognate intron
• RNA splicing happens, forming a ribonucleoprotein (RNP) holding the excised intron lariat RNA and RT
• RNPs identify DNA target sites by utilizing the RT component, and through base pairing with the intron RNA
• Reverse splicing of the intron RNA into the top strand of the double-stranded DNA occurs
• Cleavage of the bottom DNA strand by the En domain of the RT happens
• The 3' end from the cleavage acts as a primer to initiate a DNA-primed reverse transcription of the already inserted intron RNA
• The resulting intron cDNA integrates into the host genome through cellular DNA recombination or repair mechanisms
• LI.LtrB group IIA, with its Ecl5 and RmInt1 group IIB family, are targetrons
• Red markings show interactions via DNA target recognition by mobile group II introns in RNA
• Base-pairs in E1 and E2 exons, important for DNA targeting, are highlighted in purple and blue
• They are recognized by a reverse transcriptase (RT)
• CS means bottom-strand cleavage site
• IS Denotes the site of Intron insertion

TargeTron Gene Knockout System

• Plus RT, all group II intron sequences can be designed at the EBS
• EBS/"IBS" interactions enable "reverse splicing" of RNA and cDNA synthesis
• This facilitates targeted insertional mutagenesis which is mediated by RT and 3' ends of intron lariat

RNA World in a Test Tube

• In vitro RNA can be generated via gene synthesis and in vitro transcription
• In vitro RNA requires:
  • RNA polymerase
  • Promoter
  • Insert DNA
• In vitro transcription results in purified RNA transcripts
• Undergoing SELEX increases the copy number of RNA

SELEX: Systematic Evolution of Ligands by Exponential Enrichment

• This enables the in vitro selection or systematic evolution of ligands using exponential enrichment
• The starting material may be random or natural ribozyme sequences from a DNA template

Aptamers

• Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule

RNA Ligases

• Self-ligating ligases:
  • Ligases
  • Replicating ligase systems

Creation of artificial RNA

• Creation of synthetic ribozymes is possible- including self-cleaving ribozymes and RNA polymerases, including those using only few different nucleotide.
• Simple RNA sequences possess the ability to exhibit catalytic traits

Self-Replicating Ligase Ribozymes

• In a self-replicating ligase ribozyme, A plus B makes T, and T is a ribozyme/ligase (i.e., product = ribozyme)
• An RNA enzyme that catalyzed the RNA-templated joining of RNA was converted to a format whereby 2 enzymes catalyze each other's synthesis from a total of 4 oligonucleotide substrates

Features of a Replicating Ligase Ribozyme

• Self-sustained exponential amplification occurs with a doubling time of about one hour and is indefinitely continued
• These enzymes undergo self-sustained exponential amplification even in the absence of proteins or other biological materials
• Populations of various cross-replicating enzymes were constructed/ allowed to compete, which promoted recombinant replicators to arise
• These replicating RNA enzymes can serve as genetic system experimental models and allow different selective outcomes

Lehman Hypothesis-Cooperation

• 1. RNA can self-assemble (act in trans)
• 2. RNA can be catalytic.
• 3. RNA can promote ligation (self-ligation).
• 4. RNA can promote ligation and assembly of other RNA polymers (ribozymes).
• 5. RNA can promote "self-replication".
• 6. RNA = information = ribozyme = replicator = Life?

Cooperation

• Mixtures of various RNA fragments (X,W,Z,Y, derived from a group I intron) can self-assemble to self-replicating ribozymes.
• The assemblies can spontaneously form cooperative catalytic cycles and networks.
• The RNA molecules' assembly forms the basis for function as catalysts
• Functional RNA molecules can support the early development of life

Origins of Replication Building Blocks

• RNA polymers and self-templating surfaces of clays and minerals
• "Short" oligomers proceed to longer RNA strands because of ligation due to ribozymes
• Ribozymes act as building blocks (Lehman Hypothesis)

A RNA-Based World

• The RNA world hypothesis regarding the early evolution of life relies on the premise that RNA sequences can catalyze replication The ribozyme uses: -Nucleoside triphosphates -The coding information of an RNA template to extend an RNA primer
• With all factors, RNA can catalyze RNA replication

Selected Characteristics of the Tetrahymena Ribozyme RNA

• Designed for chemical ligation of RNA-binding peptides
• RNA molecule has two peptide binding sites that capture the two peptides and ligates them together