Recent advances in aptamer applications for analytical biochemistry.

Aptamers are typically defined as relatively short (20-60 nucleotides) single-stranded DNA or RNA molecules that bind with high affinity and specificity to various types of targets. Aptamers are frequently referred to as "synthetic antibodies" but are easier to obtain, less expensive to produce, and in several ways more versatile than antibodies. The beginnings of aptamers date back to 1990, and since then there has been a continual increase in aptamer publications. The intent of the present account was to focus on recent original research publications, i.e., those appearing in 2019 through April 2020, when this account was written. A Google Scholar search of this recent literature was performed for relevance-ranking of articles. New methods for selection of aptamers were not included. Nine categories of applications were organized and representative examples of each are given. Finally, an outlook is offered focusing on "faster, better, cheaper" application performance factors as key drivers for future innovations in aptamer applications.

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(R) 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.

Preparation of genome-wide DNA fragment libraries using bisulfite in polyacrylamide gel electrophoresis slices with formamide denaturation and quality control for massively parallel sequencing by oligonucleotide ligation and detection.

Bisulfite sequencing is widely used for analysis of DNA methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by polymerase chain reaction (PCR) amplification of specific regions of interest that, overall, converts C-->T (thymine) and 5mC-->C and then capillary sequencing to measure C versus T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite conversion sample preparation to include a human genome-wide fragment library for SOliD. The current article features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment library conversion, which we refer to as Bis-PAGE, capillary-based size analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library fragments. smPCR/Capillary Sanger sequencing of approximately 200 amplicons unambiguously demonstrated greater than 99% C-->T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. Although these particular Bis-PAGE conversion and quality control methods were exemplified in the context of a fragment library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.

Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARbeta2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens.

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted "gold standard" for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARbeta2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARbeta2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher's exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARbeta2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARbeta2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.

Synthesis of conformationally preorganized and cell-permeable guanidine-based gamma-peptide nucleic acids (gammaGPNAs).

A general method for preparing optically pure guanidine-based gamma-peptide nucleic acid (gammaGPNA) monomers for all four natural nucleobases (A, C, G, and T) is described. These second-generation gammaGPNAs differ from the first-generation GPNAs in that the guanidinium group is installed at the gamma- instead of the alpha-position of the N-(2-aminoethyl)glycine backbone unit. This positional switch enables GPNAs to be synthesized from relatively cheap L- as opposed to D-amino acids. Unlike their alpha-predecessors, which are randomly folded, gammaGPNAs prepared from L-amino acids are preorganized into a right-handed helix and bind to DNA and RNA with exceptionally high affinity and sequence selectivity and are readily taken up by mammalian cells.

Materializing the potential of small interfering RNA via a tumor-targeting nanodelivery system.

The field of small interfering RNA (siRNA) as potent sequence-selective inhibitors of transcription is rapidly developing. However, until now, low transfection efficiency, poor tissue penetration, and nonspecific immune stimulation by in vivo administered siRNAs have delayed their therapeutic application. Their potential as anticancer therapeutics hinges on the availability of a vehicle that can be systemically administered, safely and repeatedly, and will deliver the siRNA specifically and efficiently to the tumor, both primary tumors and metastases. We have developed a nanosized immunoliposome-based delivery complex (scL) that, when systemically administered, will preferentially target and deliver molecules useful in gene medicine, including plasmid DNA and antisense oligonucleotides, to tumor cells wherever they occur in the body. This tumor-targeting nanoparticle delivery vehicle can also deliver siRNA to both primary and metastatic disease. We have also enhanced the efficiency of this complex by the inclusion of a pH-sensitive histidine-lysine peptide in the complex (scL-HoKC) and by delivery of a modified hybrid (DNA-RNA) anti-HER-2 siRNA molecule. Scanning probe microscopy confirms that this modified complex maintains its nanoscale size. More importantly, we show that this nanoimmunoliposome anti-HER-2 siRNA complex can sensitize human tumor cells to chemotherapeutics, silence the target gene and affect its downstream pathway components in vivo, and significantly inhibit tumor growth in a pancreatic cancer model. Thus, this complex has the potential to help translate the potent effects of siRNA into a clinically viable anticancer therapeutic.

Capillary electrophoretic analysis of methylation status in CpG-rich regions by single-base extension of primers modified with N6-methoxy-2,6-diaminopurine.

Polymerase-mediated single-base extension (SBE) of primers using a fluorescently labeled 2',3'-dideoxynucleotide triphosphate terminator was originally commercialized as SNaPshot for analysis of single-nucleotide polymorphisms by capillary electrophoresis (CE). Application of this general method to bisulfite-converted/PCR-amplified genomic DNA (gDNA) to analytically infer polymorphic methylation status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich regions of gDNA has been noted previously by others to be limited by CE mobility-shifted peaks for SBE products derived from guanine (G)/adenine (A)-mixed-base primers used to hybridize to possible polymorphic sites (i.e., C vs. thymine [T] resulting from 5mC vs. C, respectively). This limitation is precluded in the current study by using novel SNaPshot primers modified with N(6)-methoxy-2,6-diaminopurine (K), which was originally described in 1991 by Brown and Lin as a unique adenine-guanine analog capable of participating in three H-bonds with C or T in DNA. Oligonucleotides modified by K as a bispecific complement for C/T are commercially available or can be readily synthesized, and they may have utility in other assay formats used to analyze CpG methylation status.

Methylation-dependent fragment separation: novel analysis of 5-methyl cytosine by capillary electrophoresis of amplified DNA using PCR incorporation of chemically modified dCTP.

Methylation of the cytosine (C) ring to form 5-methyl cytosine (MeC) in normally unmethylated CpG-rich regions of promoters in genes is associated with transcriptional silencing. Quantification of MeC is of current interest in findining new biomarkers for cancer. To this end, and for basic research in epigenomics, we have investigated a new method for relatively simple measurement of MeC content by capillary electrophoresis (CE). PCR amplicons for CE analysis are generated from bisulfite-converted DNA [C --> uracil (U)] using fluorescently labeled primers that anneal independent of methylation status. Resultant incorporation of C vs. T at original MeC vs. C positions can lead to separate CE peaks for signal integration that is proportional to MeC content. Furthermore, these PCR products are suitable for additional methylation analyses by sequencing, single-base extension, or TaqMan. Interestingly, PCR using alpha -thio-dCTP led to greater CE separations.

Tumor-targeting nanoimmunoliposome complex for short interfering RNA delivery.

The potential of short interfering RNA (siRNA) to be developed for therapeutic use against cancer depends on the availability of an efficient tumor-specific delivery vehicle. We have previously shown that a nanoscale nonviral liposome-based complex that includes an anti-transferrin receptor single-chain antibody fragment as the targeting moiety can, when systemically administered, specifically and efficiently target primary and metastatic tumors and deliver molecules useful in gene medicine, including plasmid DNA and antisense oligonucleotides. Here we explore the ability of this complex to deliver a fluorescein-labeled siRNA to tumor cells in vivo and examine the intracellular localization in vitro by confocal microscopy. We show that the immunoliposome--siRNA complex maintains its nanoscale size and, using three separate tumor models, can efficiently and specifically deliver siRNA to both primary and metastatic disease after systemic delivery, thus increasing the possibility for translating the potent effects of siRNA observed in vitro into clinically useful therapeutics.

Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2.

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 '-deoxyinosine (dI) and 2 '-O-methylribonucleotide (2 '-OMe) residues: 5'-d(TITIT)-2'OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.

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.

Cell-permeable peptide nucleic acid designed to bind to the 5'-untranslated region of E-cadherin transcript induces potent and sequence-specific antisense effects.

Establishing a general and effective method for regulating gene expression in mammalian systems is important for many aspects of biological and biomedical research. Herein we report the antisense activities of a cell-permeable, guanidine-based peptide nucleic acid (PNA) called GPNA. We show that a GPNA oligomer designed to bind to the transcriptional start-site of human E-cadherin gene induces potent and sequence-specific antisense effects and is less toxic to the cells than the corresponding PNA-polyarginine conjugate. GPNA confers its silencing effect by blocking protein translation. The findings reported in this study provide a molecular framework for designing the next generation cell-permeable nucleic acid mimics for regulating gene expression in live cells and intact organisms.

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA.

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.