Since the development of modern molecular biology, cells, and their structures have been studied in detail. It has now become apparent that highly organized molecular structures act as molecular machines or nano-machines within cells. DNA, RNA polymerase, the spliceosome, aminoacyl-tRNA synthetases, and the ribosome are examples that participate in transcription and translation of genetic information into proteins. Other examples are the flagellum, found in bacteria, the cilium, present in eukaryotic cells, and the blood clotting cascade, antibodies, myosin, kinesin, the
Figure 1: A RNA Polymerase II Transcriptional Machine.
Different views of a molecular model illustrate the architecture of the RNA polymerase II-Mediator core transcription initiation complex (4V1O).
Many innovations in synthetic chemistry and instrumentation have already enabled their study and new developments in the field are speeding up investigations of how these machines work.
Gene Knockouts using RNAi
Genes can now be knocked down or down-regulated using RNA interference (RNAi). RNAi can induce rapid degradation of the transcript of a gene by down-regulating its translation. Synthetic double-stranded RNA duplexes have been shown to be useful tools for the design of RNAi screens for the study of gene expression or gene expression pathways.
Long noncoding RNAs (LncRNAs)
Since the finish of the human genome project, now known as the postgenomic era, non-coding RNAs have been recognized as important players in cell regulation with diverse functions in health and disease. It appears that non-coding RNAs, once called “junk” RNA, when misregulated are at the cause of cancer and other metabolic diseases. For example, it was recently discovered that estrogen receptors can cause the androgen-signaling pathway to promote prostate cancer growth. The long noncoding RNA (lncRNA) NEAT1 acts as a modulator of prostate cancer. The estrogen receptor alpha (ERα) can be activated by NEAT1. NET1 was observed to be expressed in cancerous prostate cells. Recent findings indicate that deregulated lcnRNAs may impact a diverse array of human cancers and their progression. Furthermore, many scientists now suspect that lncRNAs are the missing link to cancer, especially the treatment of cancers that are refractory to classical existing therapeutics. Therefore, lncRNAs now represent new biomarkers and targets for drug development for many cancers.
Biosynthesis Inc. can synthesize long RNAs using chemical synthesis or in vitro transcription to obtain long RNA transcripts with the help of bacteriophage polymerases (T7 RNA polymerase) in high-quality.
Chromatin immunoprecipitation methods reveal existence of noncoding RNAs (ncRNAs)
Recent advances in RNA sequencing technologies in combination with chromatin immunoprecipitation (ChIP) methods have now revealed the existence of thousands of noncoding RNA species. Often, these lncRNAs are also associated with histone modifications.
Earlier studies of RNA in mammalian cells or tissue involved incorporating radioactive nucleotides into cellular RNA, extraction of the RNA, examination of the RNA for size, determination of base composition, detection of chemically modified bases, and specific measurements of the relative synthesis rate. Early on, this was done using cDNAs in a run-on analysis. This way heterogeneous nuclear RNA (hnRNA), now also called pre-mRNA, was discovered. One of the first discovered lncRNA is Xist RNA. Xist RNA is involved in X-chromosome repression in mammals, including humans.The availability of synthetic oligonucleotides, native or modified, together with many innovative improvements made during the last decades in analytical methodologies allow now studying gene expression and its modulation by targeting RNA or the genome itself. Advancements in nucleic acid chemistry drive the development of new generations of therapeutics. Artificial nucleic acids such as bridged nucleic acids (BNAs) are poised to play major roles in these developments.
Applications
Typical applications for the use of double-stranded gene fragments (ds gfDNA or gfRNA or gXNA) are:
- CRISPR/Cas9
- qPCR Controls
- Gene Construction or Assembly
- The use of genomic DNA or RNA fragments (gXNA) for the production of recombinant antibodies
- As Enzyme Substrates
- For in vitro transcription approaches
- Gene Assembly
- Affinity capture probes
CRISPR-Cas
The increase in publications using the CRISPR Cas systems indicates that this technology offers itself for the development of new innovative applications in biology and possibly in bio-medicine. The CRISPR Cas system in now used in many labs worldwide. Just recently, Kiani et al., in 2015, demonstrated that Cas9 guide RNA (gRNA) system engineering can be utilized for genome editing, activation, and repression. The altering of the length Cas9-associated guide RNA allowed the control of Cas9 nuclease activity. Also, the researchers report that they were able to perform genome editing and transcriptional regulation using a single Cas9 protein simultaneously.
CRISPRs (Clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of oligonucleotide sequences found in bacteria and archaea as a defense and regulatory mechanisms allowing them to react to various environmental stressful situations, such as a virus attack. CRISPR-Cas functions as a prokaryotic immune system that confers resistance to exogenous genetic elements such, for example from plasmids and phages, by providing a form of acquired immunity. The CRISPR Cas system has two novel features allowing the host to incorporate specifically short sequences from invading genetic elements into a region of its genome. This genomic region is distinguished by clustered regularly interspaced short palindromic repeats (CRISPRs). These sequences are transcribed and precisely processed into small RNAs to guide a multifunctional protein complex (Cas proteins) to recognize and cleave incoming foreign genetic material. Hence, exogenous DNA is cleaved into smaller pieces by the Cas proteins and inserted into CRISPR locus. When expressed, CRISPR loci guide Cas proteins to silence exogenous genetic elements.
The CRISPR Cas system is thought to be an adaptive immunity system that uses a library of small noncoding RNAs as a powerful weapon against fast-evolving viruses and as a regulatory system by the host cells.
RNA- guided endonucleases (RGENs) adapted from the CRISPR/Cas system can now be used as engineering tools. The Cas9 protein forms a sequence-specific endonuclease when in complex with two RNAs. Already, RGENs enable genome editing by delivering purified recombinant Cas9 proteins together with guide RNAs into cultured mammalian cells and fibroblasts.
CRISPR interference (CRISPRi) uses a catalytically inactive version of the Cas9 (dCas9) endonuclease and a chimeric small guide RNA (sgRNA) to bind DNA at either promoter or coding regions to regulate genes by repressing transcription initiation or elongation.
In vitro transcription templates for sgRNA can be synthesized by annealing and extension of two complementary synthetic oligonucleotides.
Already, various cloning vectors, recombinant Cas proteins, and synthetic DNA and RNA-based oligonucleotides are available as toolkits for genetic engineering. The further development together with new and improved experimental and computational methods using these systems promise to accelerate our understanding of a biological system and enable the design of new biological systems for the regulation and modulation of gene expression.
BSI can synthesize customer tailored genomic DNA or RNA fragments (gfXNAs). These are useful tools for many applications such as the one listed above.
qPCR Controls
For the validation of PCR-based or qPCR-based assays, control samples are needed. The use of proper controls allows for the validation of biochemical, biological, molecular biology, and molecular medicine techniques and methods. Controls allow verification of analytical results to ensure these are accurate and that the assays used work as indented. The following controls should are recommended for their use in PCR or qPCR reactions. Many of these can be generated synthetically.
Negative Controls
No Template controls (NTCs)
NTCs omit any DNA or RNA template from a reaction and serve as a general control for extraneous nucleic acid contamination. The detection of an amplification signal in this control sample indicates a potential contamination or the formation of primer dimers. Background PCR signals may be ignored if the difference in Cq value between the NTC and the sample with the highest Cq value is sufficiently large.
No reverse or minus reverse transcriptase control (NRT or MRT)
An NRT control is used to assure the absence of contaminating genomic DNA (gDNA) for qPCR-based gene expression analysis. However, a reverse transcription step of a qRT-PCR experiment in the absence of reverse transcriptase needs to be performed. An efficient strategy for the removal of contaminating DNA is to include an enzymatic DNase treatment step. Unfortunately, this treatment may not be 100% effective, and a careful check for the absence of DNA will be needed as well. This control helps to assess the amount of DNA contamination present in an RNA preparation.
No amplification control (NAC) or no enzyme control (NTC)
The no amplification control (NAC) omits the enzyme, the DNA polymerase, used for the assay from the PCR reaction. This control checks for background fluorescence that is not a result of the PCR. This type of fluorescence is typically caused by the use of a degraded, dual-labeled probe. However, when utilizing SYBR-Green probe chemistries, this control is unnecessary.
Positive Controls
Positive controls allow for the verification of negative amplification results. The use of appropriate positive controls in qPCR experiments is recommended to determine false positive signals.
External or exogenous positive control (EPC)
External or exogenous positive controls are one or more separate samples that contain a control template. This can be any synthetic DNA or RNA sequence.Usually, positive controls are assayed in separate reaction wells or tubes in addition to the experimental samples. EPCs serve as controls to establish that the reverse transcription and or PCR reaction conditions work optimally in the assay. External DNA or RNA positive controls can be spiked into experimental samples. If assayed in parallel or multiplex format with the targets of interest these controls allow verification whether samples contain any components that inhibit reverse transcription and or PCR.
Internal or endogenous positive controls (IPC or EPC)
Internal positive controls can be used together with templates and primers as control targets. IPCs are included in the reaction along the target of interest. The control target should be easily distinguished, for example, by electrophoretic migration or Tm, from the target of interest. The use of IPCs
Positive PCR Controls (PPCs)
Positive PCR controls can also contain a plasmid with a primer assay that allow detection of a sequence it produces thereby confirming the performance of the PCR.
DNA extraction controls (DECs) and RNA extraction controls (RECs)
In addition, DNA extraction controls (DECs) and RNA extraction controls (RECs) can be used to verify the efficiency of the PCR assay as a whole. The use of these controls more closely mimics the test samples, as compared to the other control types.
Gene Construction and Assembly
Assembly of large fragments of DNA encoding whole genes for biochemical pathways or part of a genome still presents a significant experimental challenge. Different strategies that utilize cloning The DNA assembler method introduced by Shao et al., in 2009, allow researchers to assemble DNA or gene fragments or blocks in sizes of ~9 kb up to ~19 kb. These pieces of DNA can contain up to eight genes. The one-step method uses in vivo homologous recombination in yeast (S. cerevisiae) for the assembly and integration of a biochemical pathway. This technique is thought to enable the insertion of exogenous DNA molecules into the cell to potentially establish whole, large biochemical pathways or even a whole genome. Nowadays, these techniques and methodologies are heavily used in synthetic biology.
Typically, for the assembly or the construction of genes, gene fragments are synthesized, either chemically or synthetically, or by using combinations of both methodologies. Next, the correctly assembled gene fragments are selected, and gene assemblies with deletions are removed as well as other unwanted modifications.
Finally, the sequence of the inserted fragments will need to be confirmed. In general electrophoresis methods such as polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), mass spectrometry (MS), and/or Sanger Sequencing are used.
BSI can synthesize custom gfXNAs, as dsDNA or RNA, or ssDNA or RNA fragments, which are useful tools for many applications such as the ones listed above.
Use of gene blocks (gXNA) for the production of recombinant Antibodies
Monoclonal antibodies (mAbs) are now used for many diagnostics and therapeutics. The demand for these proteins has sparked the development of large-scale manufacturing technologies to allow production in gram if not kg amounts. Viruses, yeast, or cell cultures such as Chinese hamster ovarian (CHO) cells can be used to create or engineer recombinant antibodies. Recent advances in molecular biology now allow the synthesis of antibodies de novo in vitro without the use of animals. Because complete mouse antibodies have a deleterious effect in humans, generation of humanized or fully human monoclonal antibodies is now the ultimate goal. For the production of mAbs useful for human therapeutics the following steps need to be taken in account: (1) humanization of mouse monoclonal antibodies, (2) human antibody isolation using transgenic mouse systems or models, (3) application of phage display in generating human antibodies and/or antibody fragments in vitro, and (4) production and manufacturing of humanized and/or human antibodies. Historically, transgenic mouse systems such as phage display methods were the primary technologies used for the production of recombinant mAbs. The techniques rely on rapid cloning of immunoglobulin gene segments for the creation of antibody libraries with slightly different amino acid sequences. Antibodies with desired specificities can then be selected. For the production of recombinant antibodies usually recombinant DNA sequences are translated and displayed on the surfaces of cells or phage particles. However, newer gene editing tools, such as the CRISPR-Cas system, zinc finger nucleases (ZFNs) based systems or transcription activator-like effector nucleases (TALENs), together with large synthetic DNA or RNA gene blocks have the potential to speed up this process.
Enzyme Substrates
Synthetic DNA and RNA fragments or blocks can be used to study the specificity, selectivity, and kinetics of various oligonucleotide binding enzymes that cleave or repair DNA or RNA. For example, two non-radioactive assays can be used to evaluate mispaired heteroduplex DNA cleavage. These are a PCR amplification method and an oligonucleotide-based assay. Using these methods, Langhans and Palladino in 2009 demonstrated that most restriction endonucleases are capable of site-specific double-strand cleavage when heteroduplex mispaired DNA substrates were used. They also showed that certain mispaired substrates inhibit cleavage to undetectable levels. Various forms of synthetic oligonucleotides in different sizes can be prepared as substrates for the study of DNA and RNA-binding enzymes. Synthetic gene fragments modified or non-modified can be used as single-stranded DNA or double-stranded DNA for the study of single-strand breaks, double strand breaks, base modifications and their repair. In addition, synthetic gene fragments in various sizes are useful tools for the study of replication mechanism. These can be modified as well. Various modifications can be selectively incorporated into oligonucleotide substrates. Synthetic oligonucleotides can be used as tools for the study of DNA repair diseases at the molecular level, such as amyotrophic lateral sclerosis (ALS), Ataxia, Alzheimer’s, Huntington’s disease and others.
In vitro transcription (IVT)
In vitro transcription (IVT) refers to the synthesis of RNA using a T7 promoter and purified enzymes. This procedure allows for the template-directed synthesis of RNA oligonucleotides. Short oligonucleotides and large oligonucleotides can be synthesized in µg to mg quantities. IVT allows for the synthesis of RNA in the laboratory. IVT can be used to
Bio-Synthesis provides long synthetic RNA oligonucleotides and controls using chemically synthesized RNA oligonucleotides, or Long RNA Transcription Synthesis generated from a template containing a bacteriophage promoter, a plasmid or PCR product, and cDNA.
Synthesis of long RNA by in vitro transcription is an alternative to chemically synthesized RNA. Long RNA transcripts are obtained with the help of bacteriophage polymerases (T7 RNA polymerase) in high-quality.
Affinity Capture Probes
RNA affinity capture probes can be used for the study of transcription and translation events during physiological or pathophysiological stress conditions. For example, RNA-affinity chromatography facilitates isolation and characterization of various RNA-binding proteins. In the past decades, affinity chromatography has been used to purify diverse sets of biological molecules. For example, poly-A tailed RNA-bound proteins can be isolated using poly-U Sepharose. The development of more sensitive analytical methods in more recent years now allows for the analysis and characterization of smaller quantities of biological materials isolated from cells or even in vivo studies. Hybridization capture probes that contain a conjugated biotin moiety can be used to pull out or detect specific sequence regions. Many target enrichment strategies using hybridization probes have been developed to expand sequencing technologies.
Next generation sequencing (NGS) is an emerging diagnostic tool that utilizes amplification-based target enrichment strategies for the genomic profiling of cancer tissue. Different types of capture probes have already been designed and utilized for the specific enrichment of genomic regions to allow for multiplex analysis of tens of thousands of genomic loci. Among them are annealing probes such as padlock probes, molecular inversion probes, connector inversion probes, FRET-probes, or molecular beacons, as well as others. Artificially modified oligonucleotides, for example, using bridged nucleic acids (BNAs) allow for the synthesis of dual labeled qPCR primers and RNA probes use in 5' nuclease assays. Spiking oligonucleotides with BNA base in the probes or primers increase sensitivity in real-time quantitative PCR assays. Specific BNA probes allow for distinguishing DNA base-pair mismatches with increased sensitivity and are commonly used for SNP genotyping assays.
BSI custom-synthesizes various forms of DNA or RNA oligonucleotides that are useful tools for many applications in genomics and molecular biology.
Glossary
BNA | Bridged Nucleic Acid |
Cas: | CRISPR-associated genes, located in the vicinity of CRISPR array and are necessary for the silencing of invading nucleic acid. |
Cas9t: | Cas9–crRNA–tracrRNA ternary complex: Functions as an RNA-guided DNA endonuclease and mediates site-specific DNA cleavage. |
Cas9mRNA | |
Catalytically dead Cas9 (dCas9) | dCas9 is a catalytically dead version of Cas9 endonuclease. |
Clustered regularly interspaced short palindromic repeat (CRISPR): | An array of short conserved repeat sequences interspaced by unique DNA sequences of similar size called spacers that often originate from phage or plasmid DNA. CRISPR array together with Cas genes |
CRISPR RNA (crRNA): | small RNA molecule generated by transcription and processing of the CRISPR array. crRNA is composed of a conserved repeat fragment(s) and a variable spacer sequence, which matches the |
CRISPR interference (CRISPRi) | CRISPRi uses a catalytically dead version of the Cas9 (dCas9) endonuclease and a chimeric small guide RNA (sgRNA) to bind DNA at either promoter or coding regions to regulate genes by repressing transcription initiation or elongation. |
gDNA | Genomic DNA. |
Homologous repair (HR): | Error-free DNA repair pathway that seals the broken DNA molecule using a homologous sequence (template). |
hnRNA | Heterogeneous nuclear mRNA. hnRNAs are the original RNA transcripts found in eukaryotic nuclei before post-transcriptional modifications. Many RNA types are found in the nucleus, including mRNA precursors (pre-mRNA) and other types of RNA. |
lncRNA | Long noncoding RNA. |
lncRNA database | |
mRNA | Messenger RNA |
miRNA | MicroRNA |
Non-homologous end joining (NHEJ): | A pathway that repairs DNA double strand breaks (DSB) in the absence of a homologous template; usually leads to small insertions or deletions. |
Protospacer adjacent motif (PAM): | A short conserved nucleotide stretch located in the vicinity of a |
Protospacer: | A fragment in the target DNA, which matches a spacer sequence in the CRISPR array. |
RNA- guided endonucleases (RGENs) | RGENs are adapted from the CRISPR/Cas system and are now used as genetic engineering tools. The Cas9 protein forms a sequence-specific endonuclease when in complex with two RNAs. |
Scaffold RNAs (scRNA) | ScRNAs use fusions to the dCas9 or the sgRNA to localize functional protein domains. For example, effector proteins that can additionally regulate gene expression. |
Single guide RNA (sgRNA): | RNA hairpin obtained by connecting crRNA and tracrRNA into a single molecule. |
Short guide RNA (sgRNA) | sgRNA directs the Cas9 nuclease to a cleavage site in the genome. sgRNA must match a 20 |
Single-stranded oligonucleotides (ssODNs) | ssODNs can be used as repair templates. These are usually 120- 200 |
Protospacer adjacent motif (PAM) | PAM or NGG motif essential for DNA cleavage. |
tmRNA | Transfer-messenger RNA, also known as 10Sa RNA or SsrA, is a bacterial RNA molecule having dual tRNA-like and messenger RNA-like properties. It forms an RNA-protein complex with ribosomal S1, |
RNA database | RNA central. http://rnacentral.org/ |
Transcription activator-like effector nuclease (TALEN): | An artificial nuclease obtained by fusing Xanthomonas transcription activator-like effector (TALE) DNA binding domains to the nonspecific nuclease domain. |
Trans-acting CRISPR RNA (tracrRNA): | Trans-encoded small RNA molecule that forms a duplex with a |
Triple helix forming oligonucleotide (TFO): | An artificial oligodeoxynucleotide, which binds to the polypurine sequences of the double-stranded DNA forming DNA triple helix. |
Zinc finger nuclease (ZFN): | An artificial nuclease created by fusing zinc finger motifs, which serve as DNA recognition modules, to a nonspecific DNA cleavage domain of the FokI restriction endonuclease. |
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