Protein biomarker validation via proximity ligation assays

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Sensitive protein detection via triple-binder proximity ligation assays

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.     

Protein ligation in living cells using sortase

Sortagging is a versatile method for site‐specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca²⁺‐independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C‐terminal sortase‐recognition motif (LPXTG); we used proteins with an N‐terminal (oligo)glycine as nucleophiles. We show that sortase‐dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein‐based nucleophiles in different intracellular compartments.     

DNA hybridization assays using metal-enhanced fluorescence

We describe a new approach to DNA hybridization assays using metal-enhanced fluorescence. Thiolated oligonucleotides were bound to silver particles on a glass substrate. Addition of a complementary fluorescein-labeled oligonucleotide resulted in a dramatic time-dependent 12-fold increase in fluorescence intensity during hybridization. Proximity to silver particles resulted in a decreased fluorescence lifetime. This effect is thought to be the result of enhanced fluorescence from fluorescein near metallic silver particles. Hybridization could thus be measured from the decay kinetics of the emission, which can be measured independently from the emission intensity. These results suggest the use of silver particles as a general approach to measure DNA hybridization as a method to increase the sensitivity of DNA detection.     

Artificial DNA splicing using directed ligation]

An approach to the directed genetic recombination in vitro has been devised, which allows for joining, in a predetermined chemical-enzymatic way, a series of DNA segments to give a precisely spliced polynucleotide sequence (DNA Splicing by Directed Ligation, SDL). The approach makes use of amplification, by several polymerase chain reactions (PCR), of the chosen DNA segments. The corresponding primers contain recognition sites of the class IIS restriction endonucleases, yielding protruding ends of unique primary structures. The protruding ends of the segments to be joined together are structurally predetermined to make them mutually complementary. Ligation of the mixture of the segments so synthesized gives the desired sequence in an unambiguous way. The suggested approach has been exemplified by the synthesis of a totally processed (intronless) gene encoding human mature interleukin-1 alpha.     

Protein detection using biobarcodes

Over the past 50 years the development of assays for the detection of protein analytes has been driven by continuing demands for higher levels of sensitivity and multiplexing. The result has been a progression of sandwich-type immunoassays, starting with simple radioisotopic, colorimetric, or fluorescent labeling systems to include various enzymatic or nanostructure-based signal amplification schemes, with a concomitant sensitivity increase of over 1 million fold. Multiplexing of samples and tests has been enabled by microplate and microarray platforms, respectively, or lately by various molecular barcoding systems. Two different platforms have emerged as the current front-runners by combining a nucleic acid amplification step with the standard two-sided immunoassay. In both, the captured protein analyte is replaced by a multiplicity of oligonucleotides that serve as surrogate targets. One of these platforms employs DNA or RNA polymerases for the amplification step, while detection is by fluorescence. The other is based on gold nanoparticles for both amplification as well as detection. The latter technology, now termed Biobarcode, is completely enzyme-free and offers potentially much higher multiplexing power.     

Proximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide-oligonucleotide-phycoerythrin conjugates ('burrs') that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.     

Direct detection of small RNAs using splinted ligation

This protocol describes a method for direct labeling and detection of small RNAs present in total RNA by splinted ligation. The assay uses a small RNA-specific bridge oligonucleotide to form base pairs with the small RNA and a 5'-end-radiolabeled ligation oligonucleotide. The captured small RNA is directly labeled by ligation. Detection of the labeled small RNAs is performed by denaturing gel electrophoresis and autoradiography or phosphor-imaging. This protocol has been successfully used to study expression of various classes of biological small RNAs from nanogram to microgram amounts of total RNA without an amplification step. It is significantly simpler to perform and more sensitive than either northern blotting or ribonuclease protection assays. Once the oligonucleotides have been synthesized and total RNA has been extracted, the procedure can be completed in 6 h.     

Site-specific fluorescent labeling of DNA using Staudinger ligation

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.     

Direct detection of genomic DNA with fluidic force discrimination assays

Herein, we describe the direct detection of genomic DNA using fluidic force discrimination (FFD) assays. Starting with extracted bacterial DNA, samples are fragmented by restriction enzymes or sonication, then thermocycled in the presence of blocking and labeling sequences, and finally detected with microbead-based FFD assays. Both strain and species identification of extracted Bacillus DNA have been demonstrated in <30 min, without amplification (e.g., PCR). Femtomolar assays can be achieved with this rapid and simple procedure.     

Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays

A simple enhanced chemiluminescent procedure for the quantitation of DNA hybridization to dot blots is described. The method utilizes DNA probes labeled with biotin, which are detected using a biotinylated streptavidin-horseradish peroxidase complex. The peroxidase enzyme then takes part in an enhanced chemiluminescent reaction with luminol, peroxide, and an enhancer. The method can be used to give quantitative results using a photomultiplier tube or qualitative results by recording the light emission on instant photographic film.     

On-chip oligonucleotide ligation assay using one-dimensional microfluidic beads array for the detection of low-abundant DNA point mutations

The detection of low-abundant DNA point mutations is very important for the early prediction of cancer, diagnostics of disease and clinical prognosis. In this paper, an on-chip oligonucleotide ligation approach that arrayed a series of functionalized beads in a single microfluidic channel was described for detection of low-abundant point mutations in p53 gene. This gene carried the point mutation with high diagnostic value for assessment of tumor progression and resectional borders. This work extended our prior efforts using one-dimensional (1-D) microfluidic beads array for protein and nucleic acid molecular profiling, and displayed high discrimination sensitivity to mutations detection due to the enhanced mass transport capability caused by microfluidic addressing format of beads array. As a demonstration, it was found that the on-chip beads ligation held high mutation discrimination sensitivity in 1 pM quantities at a SNR (signal-to-noise ratio) >2 using synthesized DNA oligonucleotides in accordance with target fragment. The RT-PCR products of tumor cell line A549, CNE2 and SKBr-3 were further examined to distinguish the point mutation at codon 175 of p53 gene. This approach was capable of detecting a point mutation in a p53 oncogene at a level of 1 mutant in 1000 wild-type sequences using PCR products without the need of LDR amplification. Additionally, this on-chip beads ligation approach also displayed other microfluidic-based advantages of simple handling (one sample injection per test), little reagent quantities, and low potential of contaminations.     

Improved quantitation of DNA curvature using ligation ladders.

It is often desirable to estimate accurately the local shape of DNA molecules. Such measurements are useful in understanding the intrinsic contribution of DNA sequence to curvature, as well as in assessing the effects of chemical modifications. We have been investigating the effects of asymmetric phosphate neutralization on DNA shape using the well-characterized ligation ladder approach developed by Crothers and co-workers [D.M.Crothers and J.Drak (1992) Meth. Enzymol.,212, 46-71]. This technique is remarkably sensitive to differences in DNA shape. We now report a general quantitative assay of DNA curvature that we have validated using a set of phased A(5)tract standards. This approach allows simultaneous estimation of helix axis deflection magnitude and direction when a test sequence is monitored in at least three phasings relative to a reference A(5-6)tract in short DNA duplexes. Analysis using this improved approach confirms our published data on DNA curvature due to electrostatic effects.     

DNA detection using recombination proteins

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid–based tests.     

Automated four-color DNA sequencing using primers assembled by hexamer ligation

A procedure based on the assembly of sequencing primers by hexamer ligation and then using them in automated DNA sequencing is described. This method is based on a four-color fluorescent terminator chemistry. Sequencing ladders were analyzed using an ABI 373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The best results were obtained for primers assembled by ligation of four to ten hexamers. The accuracy of the method was estimated to be 99.5% up to 400 nt of the read sequence, and somewhat lower at 400–600 nt.     

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.     

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.     

Protein homology detection using string alignment kernels

MOTIVATION: Remote homology detection between protein sequences is a central problem in computational biology. Discriminative methods involving support vector machines (SVMs) are currently the most effective methods for the problem of superfamily recognition in the Structural Classification Of Proteins (SCOP) database. The performance of SVMs depends critically on the kernel function used to quantify the similarity between sequences. RESULTS: We propose new kernels for strings adapted to biological sequences, which we call local alignment kernels. These kernels measure the similarity between two sequences by summing up scores obtained from local alignments with gaps of the sequences. When tested in combination with SVM on their ability to recognize SCOP superfamilies on a benchmark dataset, the new kernels outperform state-of-the-art methods for remote homology detection. AVAILABILITY: Software and data available upon request.