Alexandre Lebedev

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.

Enzymatic synthesis of structure-free DNA with pseudo-complementary properties

Long single-stranded DNAs and RNAs possess considerable secondary structure under conditions that support stable hybrid formation with oligonucleotides. Consequently, different oligomeric probes can hybridize to the same target with efficiencies that vary by several orders of magnitude. The ability to enzymatically generate structure-free single-stranded copies of any nucleic acid without impairing Watson–Crick base pairing to short probes would eliminate this problem and significantly improve the performance of many oligonucleotide-based applications. Synthetic nucleic acids that exhibit these properties are defined as pseudo-complementary. Previously, we described a pseudo-complementary A-T couple consisting of 2-aminoadenine (nA) and 2-thiothymine (sT) bases. The nA-sT couple is a mismatch even though nA-T and A-sT are stable base pairs. Here we show that 7-alkyl-7-deazaguanine and N4-alkylcytosine (where alkyl = methyl or ethyl) can be used in conjunction with nA and sT to render DNA largely structure-free and pseudo-complementary. The deoxynucleoside triphosphates (dNTPs) of these bases are incorporated into DNA by selected mesophilic and thermophilic DNA polymerases and the resulting primer extension products hybridize with good specificity and stability to oligonucleotide probes composed of the standard bases. Further optimization and characterization of the synthesis and properties of pseudo-complementary DNA should lead to an ideal target for use with oligonucleotide probes that are <25 nt in length.

Properties of pseudo-complementary DNA substituted with weakly pairing analogs of guanine or cytosine

A straightforward enzymatic protocol for converting regular DNA into pseudo-complementary DNA could improve the performance of oligonucleotide microarrays by generating readily hybridizable structure-free targets. Here we screened several highly destabilizing analogs of G and C for one that could be used with 2-aminoadenine (nA) and 2-thiothymine (sT) to generate structure-free DNA that is fully accessible to complementary probes. The analogs, which included bioactive bases such as 6-thioguanine (sG), 5-nitrocytosine (NitroC), 2-pyrimidinone (P; the free base of zebularine) and 6-methylfuranopyrimidinone (MefP), were prepared as dNTPs and evaluated as substrates for T7 and Phi29 DNA polymerases that lacked editor function. Pairing properties of the analogs were characterized by solution hybridization assays using modified oligonucleotides or primer extension products. P and MeP did not support robust primer extension whereas sG and NitroC did. In hybridization assays, however, sG lacked discrimination and NitroC paired too strongly to C. The dNTPs of two other base analogs, 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), exhibited the greatest promise. Either analog could be used with nA and sT to generate DNA that was nearly structure-free. Hybridization of probes to these modified DNAs will require the development of base analogs that pair strongly to NitrocH or sC.

Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation.

For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol.

651. Making the Optimal Messenger RNA for Gene Therapy Applications: Evaluation of Novel Nucleotide Modifications for Improved Activity

Recently, there has been significant interest in the use of messenger RNA (mRNA) based expression systems for gene therapy applications. Several groups have shown that mRNA is an attractive vehicle for therapeutic gene expression in mammals (Kormann et al. Nat. Biotechnol (2011) 29, 154; Kariko et al. Molecular Therapy (2012) 20, 948). mRNAs are expressed in the cytoplasm of cells which may improve expression in non-dividing cells, which are difficult to transfect. Additionally, Warren et al. demonstrated highly efficient induced pluripotent stem cell (iPS cells) generation by transfection of mRNAs encoding reprogramming factors (Warren et al. Cell Stem Cell (2010) 7, 618). The authors suggested that iPS cells generated in this manner should be safer than iPS cells derived by plasmid transfection or viral transduction since mRNA poses no risk of insertional mutagenesis and subsequent oncogenesis. In addition, transient expression from mRNA is desirable for applications such as genome editing using zinc-finger nucleases, TALENs and Cas9/CRISPR. Lastly, there is considerable interest in using mRNA for vaccines (Geall et al. Expert Rev Vaccines (2015) 14, 151).A key insight for the development of mRNA expression systems was the recognition that mRNA induces innate immune responses in transfected cells. Kariko et al. showed that substitution of uridine and cytidine residues with pseudouridine and 5-methylcytidine dramatically reduced innate immune recognition of mRNA (Kariko et al. Molecular Therapy (2008) 16, 1833). They also showed that pseudouridine modified RNA was translated more efficiently.These studies highlight the importance of the development of stable, non immunogenic mRNA. Activity and immunogenicity of mRNAs likely depends on the chemical modification pattern, the route of delivery and cell type or tissue transfected. To date, however, there have been few studies to assess novel chemically modifications of mRNAs. Here we synthesized a panel of numerous novel nucleotide triphosphates (NTPs). The panel consisted primarily of 5-position-modified pyrimidines but also included other pyrimidine and purine NTPs. A large number of combinations of the modifications were used to synthesize GFP and luciferase mRNAs in order to evaluate the ability of different combinations of modifications to support transcription by T7 RNA polymerase. The translation potential of the mRNAs was evaluated in rabbit reticulocyte lysates. The activity and toxicity profile of these mRNA was further evaluated in panels of different primary and immortalized cell lines. Comparison of in vitro translation with cell culture expression allows us uncouple translation from cytotoxicity, RNA stability and innate immune stimulation in cells. While trends were seen, cell type specific differences in expression were also observed. These studies greatly expand our knowledge of the optimal chemical modification of mRNA required to achieve maximal expression in different cell types.