SDS‐PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs

SDS‐PAGE is considered to be a universal method for size‐based separation and analysis of proteins. In this study, we applied the principle of SDS‐PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, – phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.     

Cyanuryl peptide nucleic acid: synthesis and DNA complexation properties

The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-aminoethyl)-N′-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids when the cyanuryl unit was located in the middle of the PNA oligomer.     

Synthesis and polymerisation of lipophilic peptide nucleic acids derived from stearic acid and pentacosa-10,12-diynoic acid

The adenine, cytosine and thymine peptide nucleic acid (PNA) monomers and PNA T₁₀ oligomers bearing either a diacetylenic or stearoyl moiety at the N- or C-terminus have been successfully prepared. The resulting thymine monomeric and T₁₀-mer derivatives have been subsequently incorporated into polydiacetylene-containing liposomes.     

The staining efficiency of cyanine dyes for single‐stranded DNA is enormously dependent on nucleotide composition

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson‐Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.     

Classifying Human Haptoglobin Phenotypes on Native‐PAGE Using a Modified Coomassie Brilliant Blue R 250 Staining

There are three types of human Haptoglobin, Hp 1‐1, Hp 2‐1, and Hp 2‐2, each characterized by a distinct combination of the two α chains of the holoprotein. A modified Coomassie Brilliant Blue R 250 (mCBB‐R250) staining method for detecting the phenotypes of human blood haptoglobin molecules is presented. Addition of excess hemoglobin to the sample allowed specific formation of different haptoglobin‐hemoglobin complexes, which, in turn can be separated into distinctive migration pattern on native‐PAGE and stained by CBB‐R250. The typing results are consistent with that using Western blotting. In comparison to the existing methods for haptoglobin typing, our method is comparable in accuracy, and is easier to carry out. The results on typing of 29 plasma samples from Taipei Blood Center were also presented.     

Lipophilic peptide nucleic acids containing a 1,3-diyne function: synthesis, characterization and production of derived polydiacetylene liposomes

Adenine-, cytosine- and thymine-containing peptide nucleic acid (PNA) monomers have been synthesized in which either diacetylenic or stearoyl moieties are attached to the N-or C-terminus; the diacetylenic group is embedded within a long hydrocarbon chain. A range of analogous lipophilic functionalized PNA oligomers have been prepared using either solid phase synthesis or a post-synthetic solution phase procedure following cleavage of the PNA oligomer from the solid support. Selected functionalized PNA monomers and oligomers have been incorporated into liposomal polydiacetylenes and characterized by UV-vis absorption spectroscopy. Preliminary investigations show that blue PDA-liposomes containing thymine-based PNAs can be formed and that production of liposomes with other PNA systems are viable.     

Understanding anomalous mobility of proteins on SDS‐PAGE with special reference to the highly acidic extracellular domains of human E‐ and N‐cadherins

During SDS‐PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ～110 residues‐long, sequence‐homologous, structurally‐analogous, extracellular domains of human E‐ and N‐cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively‐charged and positively‐charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater‐than‐expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS‐protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.     

Chimeric peptide nucleic acids incorporating (2S,5R)-aminoethyl pipecolyl units: synthesis and DNA binding studies

The design and facile synthesis of a novel chiral six-membered PNA analogue (2S,5R )-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic acid, aepipPNA, that upon incorporation into PNA sequences effected stabilization of complexes with target complementary DNA. This is the first example where a six membered-PNA is shown to be capable of forming stable complexes with DNA and further expands the repertoire of cyclic PNA analogues.     

Synthesis of novel analogs of aromatic peptide nucleic acids (APNAs) with modified conformational and electrostatic properties

Aromatic peptide nucleic acid analogs having an N-(2-aminobenzyl)glycine backbone (APNA 1) were previously identified as promising new leads for the design of polyaromatic DNA mimics. Structural modifications of 1, which lock the aromatic backbone into a unique conformation, while maintaining the same space distribution between the nucleobases as in 1, were investigated. The electrostatic potential of the aromatic backbone was also modified in an attempt to improve the solubility of these compounds in aqueous media and to evaluate how the quadrapole of the aromatic backbone may influence the biophysical properties of the APNA oligomers. PNA hexamers containing a single monomer insert of each new APNA monomer were used to explore the hybridization properties of these analogs with poly rA and poly dA. Preliminary results indicated that these modifications do not seriously alter the molecular recognition properties of APNAs towards DNA and RNA.     

PNA encoding (PNA=peptide nucleic acid): from solution-based libraries to organized microarrays

Microarray-based technologies have attracted attention in chemical biology by virtue of their miniaturized format, which is well suited to probe ligand-protein interactions or investigate enzymatic activity in complex biological mixtures. A number of research groups have reported the preparation of surfaces on microarrays with specific functional groups to chemoselectively attach small molecules from libraries. We have developed an alternative method whereby libraries are encoded with peptide nucleic acid (PNA), such that libraries which exist as mixtures in solution self-assemble into an organized microarray through hybridization to produce readily available DNA arrays. This allows libraries synthesized by split and mix methods to be decoded in a single step. An asset of this method compared to direct spotting is that libraries can be used in solution for bioassays prior to self-assembly into the microarray format.     

Synthesis and oligodeoxynucleotide binding properties of pyrrolidinyl peptide nucleic acids bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones

A series of novel conformationally rigid pyrrolidinyl peptide nucleic acids (PNA) based on d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones has been synthesized. Investigation of the binding properties of four stereoisomeric PNAs possessing different stereochemistry at the ACPC part with DNA revealed that a precise stereochemistry of the backbone is very important in determining the binding properties. Only the PNA containing (1S,2S)-ACPC can form a very stable 1:1 complex with the complementary DNA in a sequence-specific manner.     

Separation and recovery of nucleic acids with improved biological activity by acid‐degradable polyacrylamide gel electrophoresis

One of the fundamental challenges in studying biomacromolecules (e.g. nucleic acids and proteins) and their complexes in a biological system is isolating them in their structurally and functionally intact forms. Electrophoresis offers convenient and efficient separation and analysis of biomacromolecules but recovery of separated biomacromolecules is a significant challenge. In this study, DNAs of various sizes were separated by electrophoresis in an acid‐degradable polyacrylamide gel. Almost 100% of the nucleic acids were recovered after the identified gel bands were hydrolyzed under a mildly acidic condition and purified using anion exchange resin. Further concentration by centrifugal filtration and a second purification using ion exchange column chromatography yielded 44–84% of DNA. The second conventional (non‐degradable) gel electrophoresis confirmed that the nucleic acids recovered from acid‐degradable gel bands preserved their electrophoretic properties through acidic gel hydrolysis, purification, and concentration processes. The plasmid DNA recovered from acid‐degradable gel transfected cells significantly more efficiently than the starting plasmid DNA (i.e. improved biological activity via acid‐degradable PAGE). Separation of other types of nucleic acids such as small interfering RNA using this convenient and efficient technique was also demonstrated.     

Urine protein quantification in stacking gel by SDS‐PAGE

Urine total protein concentration is usually measured by the pyrogallol red‐molybdate (PRM) assay in clinical laboratories, but it is often subject to sample interference. Here, we introduce a stacking gel‐based method for accurate protein quantitation. In this method, the urine protein samples are run into the stacking gel by SDS‐PAGE where it is concentrated into a single band, and then quickly stained by 0.001% Coomassie at high temperature. High correlations were found between the BSA and urine protein standards (R² = 0.997 and 0.990, respectively). Addition of 80 ng urine protein standard into each of the ten clinical urine specimens with questionable PRM results yielded the expected increase in the results by this method. Comparison of the PRM method and with the gel quantitation approach on about 60 clinical urine samples demonstrated a general consistency between the results (R² = 0.825), but in PRM samples with lower protein concentration showed more variations. Overall, the stacking gel method might be a good alternative for clinical urine samples with suspicious protein concentration results.     

A nonradioactive method for detecting phosphates and polyphosphates separated by PAGE

Nonradioactive polyphosphate (poly(P); (PO(3) (-))(n)) species resolved by PAGE can be detected by hydrolytic degradation of the polyphosphates into orthophosphates (P(i)) with a 5 M HCl solution saturated with NaCl, followed by staining the P(i) degradation products in a 1 M HCl solution of 0.25% w/v methyl green and 1% w/v ammonium molybdate. This method detects down to 0.5 nmol of phosphate as P(i), linear poly(P) (condensed phosphate), pyrophosphate (P(2)O(7) (4) (-)), or cyclic trimetaphosphate ion (P(3)O(9))(3) (-) species. This method improves the current method of staining linear poly(P) longer than four phosphate units with Toludine blue-O after PAGE. This study also shows that Stains-All can visualize resolved linear poly(P) shorter than those visualized by Toluidine blue-O. It is hoped that this sequential hydrolytic degradation and phosphate visualization method for detecting ortho-, linear, and cyclic poly(P) species will be a useful tool, as poly(P) are being discovered in a wide variety of biological systems, and their biochemical roles are still largely unknown.     

Enrichment of low-abundant serum proteins by albumin/immunoglobulin G immunoaffinity depletion under partly denaturing conditions

We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.     

Convergent synthesis of peptide nucleic acids by native chemical ligation

[reaction: see text] A convergent strategy for synthesizing long contiguous PNA by a native chemical ligation-like technique of PNA segment couplings is presented. This approach required the synthesis of a new PNA-monomer featuring a 1-amino-2-thiol group. It is shown that the additional mercaptomethyl group leaves the hybridization properties of PNA ligation products unaffected. Furthermore, rapid and efficient fluorescence labeling of the ligation products is demonstrated.     

Microfluidic chip of fast DNA hybridization using denaturing and motion of nucleic acids

This study presents the effect of fluidic temperatures and velocities on improving DNA hybridization. The efficiency of hybridization could be improved by introducing elevated temperature in the hot region and velocity in the cold region. Compared with the conventional methods, this hybridization microchip was able to increase the hybridization signal 4.6-fold within 30 min. The 1.4-kb single-stranded target DNA was tested. The increasing tendency of the fluorescence intensity was apparent when the temperature was higher than 82 degrees C, and the fluorescence intensity reached an asymptotic value at T>90 degrees C. A mathematical model was proposed to relate the fluorescence intensity of DNA hybridization with the hot-region temperature and the cold-region velocity. Based on these results, the new hybridization chip with the processes of temperature and velocity differences will improve efficiency of DNA detection. The microchip combined with hot-region temperature and cold-region bulk flow velocity effects could provide additional efficiency in DNA hybridization.     

Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation

PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.     

Preparative alkaline urea gradient PAGE: application to purification of extraordinarily-stable disulfide-linked homodimer of human growth hormone

The 40-60 pituitary human growth hormone (hGH) isoforms are so similar in their physico-chemical properties (charge, size, hydrophobicity) that the limited resolutions of chromatographic separation methodologies have not permitted most of them to be isolated. However, application of high-resolution preparative alkaline urea gradient PAGE has facilitated isolation of a disulfide-linked mercaptoethanol-resistant (MER) 45 kDa hGH dimer. Human pituitary extracts were separated by Sephadex G-100 chromatography under alkaline conditions. Pooled fractions containing MER-45 kDa hGH, as determined by SDS-PAGE, were then separated by Sephadex G-100 chromatography under acidic conditions followed by diethylaminoethyl (DEAE) anion-exchange chromatography. Pooled DEAE fractions containing MER-45 kDa hGH and other hGH isoforms were then separated by preparative electrophoresis in an alkaline polyacrylamide gradient (5-20%) slab gel containing 8 M urea into five distinct protein zones. One electroeluted zone contained pure MER-45 kDa hGH. The dimeric hGH isoform was immunoreactive at low concentrations (effective dose to produce 50% response (ED(50)) +/- S.E. = 58 +/- 5.00 pM) in a hGH radioimmunoassay, similar to that of standard monomeric hGH (ED(50) +/- S.E. = 22.93 +/- 3.90 pM), indicating that is was conformationally intact. Alkaline urea gradient PAGE is a valuable tool for preparative separation of structurally similar proteins such as isoforms of the hGH family.     

Scanning the beta-globin gene for mutations in large populations by denaturing capillary and gel electrophoresis

Separation of mutant from nonmutant DNA sequences of 100 bp may be accomplished by using defined denaturing conditions of chemical denaturant and/or elevated temperature during electrophoresis on either polyacrylamide slab gels (denaturing gradient gel electrophoresis, DGGE) or capillary gels (constant denaturant capillary electrophoresis, CDCE). In analysis of mutant directly from a polymerase chain reaction (PCR) product mixture, both have detection sensitivities of approximately 1%. CDCE that facilitates an intermediate mutant enrichment step permits detection of mutants at fractions as low as 2 x 10(-6). Here we report the successful application of both approaches to scan for mutations of the human beta-globin gene (HBB) in two human population samples of approximately 5000 persons in the HBB. Using DGGE, the coding region and flanking intronic splice sites of HBB were scanned in a population of 4949 Han Chinese individuals in pool sizes of 48 individual DNA samples. Four point mutations ranging in mutant frequency from 0.5 to 0.0002 were identified. Using CDCE with a mutant enrichment step, these same sequences were scanned in a population of 5028, predominantly African-American juveniles (<9 years) as a single pooled DNA sample. Three point mutations were identified ranging in mutant frequency from 0.13 to 0.0005. This study shows that both the DGGE/small pool and the CDCE/large pool approaches offer the means to define the fine structure map of genetic variation in large population samples, and with appropriately engineered facilities to provide high throughput, should be useful in pangenomic scans to discover genes carrying casual mutations for common diseases.