Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.     

Innovative approaches to the use of polyamines for DNA nanoparticle preparation for gene therapy

Advances in genomic technologies, such as next generation sequencing and disease specific gene targeting through anti-sense, anti-gene, siRNA and microRNA approaches require the transport of nucleic acid drugs through the cell membrane. Membrane transport of DNA/RNA drugs is an inefficient process, and the mechanism(s) by which this process occurs is not clear. A pre-requisite for effective transport of DNA and RNA in cells is their condensation to nanoparticles of ~100 nm size. Although viral vectors are effective in gene therapy, the immune response elicited by viral proteins poses a major challenge. Multivalent cations, such as natural polyamines are excellent promoters of DNA/RNA condensation to nanoparticles. During the past 20 years, our laboratory has synthesized and tested several analogs of the natural polyamine, spermine, for their efficacy to provoke DNA condensation to nanoparticles. We determined the thermodynamics of polyamine-mediated DNA condensation, measured the structural specificity effects of polyamine analogs in facilitating the cellular uptake of oligonucleotides, and evaluated the gene silencing activity of DNA nanoparticles in breast cancer cells. Polyamine-complexed oligonucleotides showed a synergistic effect on target gene inhibition at the mRNA level compared to the use of polyamines and oligonucleotides as single agents. Ionic and structural specificity effects were evident in DNA condensation and cellular transportation effects of polyamines. In condensed DNA structures, correlation exists between the attractive and repulsive forces with structurally different polyamines and cobalt hexamine, indicating the existence of a common force in stabilizing the condensed structures. Future studies aimed at defining the mechanism(s) of DNA compaction and structural features of DNA nanoparticles might aid in the development of novel gene delivery vehicles.     

Antisense recognition of the HER-2 mRNA: effects of phosphorothioate substitution and polyamines on DNA.RNA, RNA.RNA, and DNA.DNA duplex stability

The HER-2 gene is overexpressed in a subset of breast, ovarian, lung, and pancreatic cancers. Antisense oligonucleotides suppress gene expression depending on the stability of the DNA.RNA hybrids formed at the target site. Polyamines, the cellular cations that interact with DNA and RNA, may influence hybrid stability in the cell. Therefore, we studied the ability of natural polyamines (putrescine, spermidine, and spermine) and a series of their structural analogues to stabilize DNA.RNA and RNA.RNA duplexes using melting temperature (T(m)) measurements and circular dichroism (CD) spectroscopy. Phosphodiester (PO) and phosphorothioate (PS) oligonucleotides (ODNs) (15 nucleotides, 5'-CTCCATGGTGCTCAC-3') targeted to the initiation codon region of the HER-2 mRNA, and complementary RNA and DNA ODNs, were used in this study. The relative order of thermal stability was as follows: RNA.RNA > PO-DNA.RNA > PO-DNA.PO-DNA > PS-DNA.RNA > PS-DNA.PO-DNA > PS-DNA.PS-DNA. The ability of polyamines to stabilize the duplexes improved with the cationicity of the polyamine, with hexamines being more effective than pentamines, which in turn were more effective than tetramines and triamines. However, chemical structural effects were clearly evident with isovalent homologues of spermidine and spermine. CD spectra showed B and A conformations, respectively, for the DNA and RNA helices. DNA.RNA hybrids adopted an intermediate structure between the B and A forms. These data help us to understand the role of endogenous polyamines in DNA.RNA hybrid stabilization, and provide information for designing novel polyamines to facilitate the use of antisense ODNs for controlling HER-2 gene expression.     

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.     

Antisense technology in molecular and cellular bioengineering

Antisense technology is finding increasing application not only in clinical development, but also for cellular engineering. Several types of antisense methods (e.g. antisense oligonucleotides, antisense RNA and small interfering RNA) can be used to inhibit the expression of a target gene. These antisense methods are being used as part of metabolic engineering strategies to downregulate enzymes controlling undesired pathways with regard to product formation. In addition, they are beginning to be utilized to control cell phenotype in tissue engineering constructs. As improved methods for antisense effects that can be externally regulated emerge, these approaches are likely to find increased application in cellular engineering applications.     

A plausible mechanism for gene correction by chimeric oligonucleotides

Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.     

Locked Nucleic Acids: Promising Nucleic Acid Analogs for Therapeutic Applications

Locked Nucleic Acid (LNA) is a unique nucleic‐acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid‐based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.     

Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.     

High-efficiency transfection of mammalian neurons via nucleofection

Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.     

The potential of oligonucleotides for therapeutic applications

Viral-derived particles have been widely used and described in gene therapy clinical trials. Although substantial results have been achieved, major safety issues have also arisen. For more than a decade, oligonucleotides have been seen as an alternative to gene complementation by viral vectors or DNA plasmids, either to correct the genetic defect or to silence gene expression. The development of RNA interference has strengthened the potential of this approach. Recent clinical trials have also tested the ability of aptamer molecules and decoy oligonucleotides to sequestrate pathogenic proteins. Here, we review the potential of oligonucleotides in gene therapy, outline what has already been accomplished, and consider what remains to be done.     

Microarrays for identifying binding sites and probing structure of RNAs

Oligonucleotide microarrays are widely used in various biological studies. In this review, application of oligonucleotide microarrays for identifying binding sites and probing structure of RNAs is described. Deep sequencing allows fast determination of DNA and RNA sequence. High-throughput methods for determination of secondary structures of RNAs have also been developed. Those methods, however, do not reveal binding sites for oligonucleotides. In contrast, microarrays directly determine binding sites while also providing structural insights. Microarray mapping can be used over a wide range of experimental conditions, including temperature, pH, various cations at different concentrations and the presence of other molecules. Moreover, it is possible to make universal microarrays suitable for investigations of many different RNAs, and readout of results is rapid. Thus, microarrays are used to provide insight into oligonucleotide sequences potentially able to interfere with biological function. Better understanding of structure–function relationships of RNA can be facilitated by using microarrays to find RNA regions capable to bind oligonucleotides. That information is extremely important to design optimal sequences for antisense oligonucleotides and siRNA because both bind to single-stranded regions of target RNAs.     

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2′-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.     

Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes

Mouse oocytes have proven useful in experiments aimed at studying gene function. They have been used to analyze the gain-of-function acquired after microinjection of RNA transcribed in vitro from specific gene constructs, and for establishing loss-of-function mutation obtained by injecting in vitro transcribed antisense RNA and/or synthetic oligonucleotides. This article presents protocols utilized in these studies. Specifically, the acquisition of mouse oocytes and/or embryos, the genesis of the necessary DNA and/or RNA to be used, and procedures for microinjection.     

Automated parallel synthesis of 5′-triphosphate oligonucleotides and preparation of chemically modified 5′-triphosphate small interfering RNA

A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5′-triphosphate and 5′-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5′-triphosphates and 5′-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5′-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5′-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5′-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform.     

Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.