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Functional nucleic acids as antibody alternatives for small molecule detection

IDT gBlocks® Gene Fragments and oligos facilitate 2015 iGEM Team Heidelberg’s innovations

2015 iGEM Team Heidelberg

While the University of Heidelberg has a successful history at the annual iGEM (International Genetically Engineered Machine) competition, deciding on a project—one that used synthetic biology to provide positive contributions to society, and could be completed in a 3 month summer research period, proved difficult for this year’s young team members. In Heidelberg, the newly formed iGEM team usually spends the first few months of the year researching ideas for projects. While many such ideas were discussed by the 10-member team, none of the initial project ideas were a good fit for their diverse interdisciplinary abilities. But when they finally proposed to demonstrate the broad potential applications of functional nucleic acids, team members were enthusiastic and quickly committed to the project.

The Heidelberg team adapted functional nucleic acids such as ribozymes, DNAzymes, aptamers, and detection probes to improve on several existing basic research tools, as well as to solve some real world problems. They took advantage of IDT’s gift to iGEM teams of up to 20 kb of free gBlocks® Gene Fragments, and also obtained DNA oligos and numerous base modifications. gBlocks fragments were used as templates to transcribe functional RNAs. Functional DNAs were obtained as single-stranded oligonucleotides, and modified bases were used in click chemistry reactions to visualize specific regions of a functional DNA molecule in an assay. Here is an overview of several of the team’s accomplishments.

A faster, more affordable SELEX alternative

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is the current method for selecting high-affinity, ligand-specific nucleic acids from a random library [1,2]. However, the SELEX process relies on careful planning and is time consuming, requiring multiple weeks for generation of useful aptamers and ribozymes. Furthermore, success is greatly dependent on the quality of the nucleic acid library employed in the process.

For their iGEM project, Team Heidelberg developed a novel, open-source software that generates similar high-affinity, ligand-specific nucleic acids for the detection of small molecules present in trace amounts. The algorithm they used is based on iteration of sampling and ranking sequences, and they named the software MAWS, for “Making Aptamers Without SELEX”.

Using MAWS, the students designed aptamers for several protein targets, including actin, p53, and Cas9, as well as the small molecule targets, kanamycin and ketamine. They also computed an aptamer for xylanase to support the current research of a member of Team Heidelberg 2014. At first, calculating each aptamer took several days. After tedious optimization, however, the team was able to reduce the calculation time to just a few hours.

The DNA aptamer sequence designs were ordered as single-stranded DNA oligonucleotides from IDT. Upon receipt, the team resuspended the oligos and heated them to ~90°C. The sequences were then slowly cooled, which allowed them to fold and acquire the necessary functional structure. The Heidelberg team tested these molecules using a chemiluminescence-based assay that the students developed. Through molecular dynamics simulations in silico, they demonstrated that the MAWS designed aptamers tightly bind their targets.

AptaBodies—an inexpensive, quickly generated antibody alternative

The team also designed fusion molecules that contained one to several aptamers combined with a “read out module”—in this case an HRP-mimicking DNAzyme. These “AptaBodies” (Figure 1) allowed the team to further validate their aptamers.

In what the team declares their most intriguing and possibly most widely applicable achievement, they used aptamers against protein targets in a modified western blot. Here, the AptaBody form replaced a conventional antibody to successfully detect novel target proteins. MAWS-generated AptaBodies drastically reduce the cost and time necessary for generating specific antibodies. They also make it possible to target proteins and other small molecules for which antibodies are not available, thus providing a tool that could be quite valuable in current research.

Figure 1. AptaBody design. AptaBodies consist of an aptamer, designed to bind a specific protein of interest, and a poly-A linker that couples the aptamer to an HRP-mimicking DNAzyme, forming a G-quadruplex.

Aptazymes, nucleic-acid–based circuits, and drug detection

The Heidelberg iGEM team also developed an additional software program, “JAWS” (Joining Aptamers Without SELEX), to design aptamers that could function as nucleic acid-based circuits. These molecules would be able to respond to external stimuli, with their catalytic activity activated by either the presence or absence of the cognate ligand. Such types of fusions are known as “Aptazymes” and can be applied as small molecule sensors (Figure 2).

Figure 2. Aptazyme activation by small target molecules.

As a proof of concept, Team Heidelberg developed a colorimetric aptazyme activated by the presence of ketamine. Ketamine is an anesthetic that induces sedation and memory loss, and while it has important medical applications, it is known to be frequently abused as a date rape drug. Thus, this assay could, in principle, be used as a quick test to detect date rape drugs on location.

Twin ribozyme approach to correct mutated cystic fibrosis mRNA

People afflicted with cystic fibrosis possess mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which results in the thick build-up of mucus in the lungs. This compromises lung function and usually shortens the lives of those with the disease. The Heidelberg iGEM team wanted to use functional nucleic acids to reduce symptoms of this disease. Towards this goal, they designed ribozymes to repair the most common CFTR mutation—the deletion of phenylalanine 508—through mRNA editing.

The students redesigned a series of twin ribozymes, or “twinzymes”, to excise and replace the affected sequence within the CFTR mRNA. Their experimental design joins a twinzyme along with a repair oligonucleotide, into a ribozyme assembly vector containing a Hammerhead–twinzyme–HDV (Hepatitus Delta Virus) ribozyme cassette and various numbers of Hammerhead–repair oligonucleotide–HDV cassettes, all within what will be transcribed as a single RNA. Upon transcription, the twinzyme and repair oligonucleotides should be released from the RNA by the self-cleaving activity of the Hammerhead and HDV ribozymes, making them available to then repair the mutated CFTR mRNA.

Figure 3. Twinzyme cleavage and replacement of mutated CFTR mRNA sequence. The reaction consists of 3 steps: 1) In the top panel, the twinzyme (green) recognizes and binds the mutated CFTR target mRNA sequence (blue); 2) The twinzyme then cleaves the target site at 2 locations (black arrow heads), releasing the mutated region of the mRNA; 3) Finally, as shown in the bottom panel, the twinzyme binds the repair oligonucleotide (red) that includes the missing bases encoding phenylalanine, and inserts it into the target mRNA.

The Heidelberg team used several methods to assay whether such a twinzyme design was able to both excise a targeted sequence and insert a repair oligonucleotide. They screened for twinzyme activity in yeast using fused fluorescent GFP/mCherry reporters, which, along with the ribozymes from yeast centromeric plasmids, were expressed under the control of a strong promoter. Activity was assessed by high-throughput flow cytometry. Their results suggested that the twinzyme design cleaves its targeted mRNA. Next generation RNA sequencing of samples from yeast co-expressing the fluorescent reporters and one of the constructs again indicated ribozyme cleavage activity, but showed no evidence of insertion reactions.

The precise excision of targeted mRNA sequences, demonstrated in vivo, is a notable accomplishment. Compared to existing viral and non-viral therapies currently used to treat cystic fibrosis, this ribozymal mRNA-editing approach could have significant benefits as a method of gene therapy. It is also potentially applicable in many other situations where mRNA repair could result in a functional protein. Like non-viral therapy, this method could avoid problems with immune system reaction by using liposome or nanoparticle delivery, as has been shown with siRNA [3,4]. In addition, RNA therapy, which can be applied transiently and without the need for viral-vectors, circumvents the risks still associated with DNA gene editing, such as lasting off-target effects and genetic instability. Thus, the team recommends pursuing this approach with further modified ribozyme constructs.

Recognition

The team members we interviewed, Frieda Anna Sorgenfrei and Tim Treis, agreed that the best validation of the team’s achievements was winning the coveted iGEMers Prize, which is voted on by all 259 teams at the iGEM Jamboree (September 2015, Boston, MA, USA). The award is given to the team believed to be most deserving of the Grand Prize. While the iGEM judges ultimately voted them Second Runner Up to the Grand Prize, the Heidelberg team did win Best Foundational Advance and Best Poster, and was among the small set of teams nominated for Best Software Tool, Best Applied Design, and Best Wiki. Learn more about the Heidelberg Team’s project on their Wiki, http://2015.igem.org/Team:Heidelberg/Overview.

References

  1. Ellington AD, Szostak JW. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature, 346:818–822.
  2. Tuerk C, Gold L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science, 249:505–510.
  3. Alton EW, Armstrong DK, et al. (2015) Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: A randomised, double-blind, placebo-controlled, phase 2b trial. The Lancet Respiratory Medicine, 3:684–691.
  4. Sioud M, Soerensen DR. (2003) Cationic liposome-mediated delivery of siRNAs in adult mice.Biochem Biophys Res Commun, 312:1220–1225.

Product focus—Custom dsDNA, oligos and primers, design tools

Custom dsDNA fragments

gBlocks® Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–2000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.

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Custom oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides/primers, and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

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Free online tools for oligonucleotide analysis and primer design

Explore IDT's free, online software for oligonucleotide analysis and for qPCR probe and assay design. The design engines for these tools use sophisticated algorithms that take into account factors such as nearest-neighbor analysis and buffer conditions to calculate Tm.

Related reading

iGEM teams engineer success with gBlocks® Gene Fragments—By August, 2015, this year’s iGEM teams had made significant progress on their projects. We spoke with some of the teams to find out how the competition was going, and how they were using gBlocks Gene Fragments.

iGEM students engineer biological tools for a better world—Projects from 2 of the prize-winning 2013 iGEM teams show how non-standard natural and synthetic amino acids can be used in 1) peptide synthesis and 2) tuberculosis monitoring and treatment. Both projects make use of gBlocks® Gene Fragments to speed construct assembly.

The gene construction revolution—See how use of high-quality, custom dsDNA fragments as a starting material allows you to turn what might otherwise be multi-step cloning assemblies into simpler reactions. You can often just order an entire target sequence that is ready for cloning or other uses.

Codon optimization tool makes synthetic genes easy—Use the IDT Codon Optimization Tool to simplify designing synthetic genes and gBlocks® Gene Fragments for expression in a variety of organisms.

IDT has improved the efficiency of CRISPR-Cas9 genome editing—Webinar summary: Learn how research conducted at IDT led to the development of a potent new set of CRISPR-Cas9 genome editing tools.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

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