Microfluidic isolation of aptamers with affinity towards multiple myeloma monoclonal immunoglobulins.

Multiple myeloma (MM) is a bone marrow cancer of resident plasma cells that affects 125,000 patients in the U.S. with about 30,000 new cases per year. Its signature is the clonal proliferation of a single plasma cell that secretes a patient specific monoclonal immunoglobulin (M-Ig). Targeting the M-Ig in patient serum could allow sensitive and noninvasive identification of minimal residual disease in multiple myeloma. Aptamers, which are single-stranded oligonucleotides with affinity and specificity to a target molecule, have recently been introduced as affinity reagents for recognition of MM M-Igs. Here we exploit microfluidic SELEX technology to enable rapid and efficient generation of aptamers against M-Ig proteins from MM patients. We first characterize the technology by isolating aptamers with affinity towards the monoclonal antibody rituximab as a model M-Ig and then apply the technology to isolating aptamers specifically targeting M-Igs obtained from serum samples of MM patients. We demonstrate that high-affinity DNA aptamers (KD < 50 nM) for M-Ig proteins from a patient sample could be isolated via microfluidic SELEX within approximately 12 h and using less than 100 micrograms of patient M-Ig. Such aptamers can potentially be used in personalized monitoring of minimal residual disease in MM patients.

Personalized immunoglobulin aptamers for detection of multiple myeloma minimal residual disease in serum.

Multiple myeloma (MM) is a neoplasm of plasma cells that secrete patient specific monoclonal immunoglobulins. A recognized problem in MM treatment is the early recognition of minimal residual disease (MRD), the major cause of relapse. Current MRD detection methods (multiparameter flow cytometry and next generation sequencing) are based on the analysis of bone marrow plasma cells. Both methods cannot detect extramedullary disease and are unsuitable for serial measurements. We describe the methodology to generate high affinity DNA aptamers that are specific to a patient's monoclonal Fab region. Such aptamers are 2000-fold more sensitive than immunofixation electrophoresis and enabled detection and quantification of MRD in serum when conventional MRD methods assessed complete remission. The aptamer isolation process that requires small volumes of serum is automatable, and Fab specific aptamers are adaptable to multiple diagnostic formats including point-of-care devices.

INTEGRATED MICROFLUIDIC SELEX USING FREE SOLUTION ELECTROKINETICS.

Systematic evolution of ligands by exponential enrichment (SELEX) offers a powerful method to isolate affinity oligonucleotides known as aptamers, which can then be used in a wide range of applications from drug delivery to biosensing. However, conventional SELEX methods rely on labor intensive and time consuming benchtop operations. A simplified microfluidic approach is presented which allows integration of the affinity selection and amplification stages of SELEX for the isolation of target-binding oligonucleotides by combining bead-based biochemical reactions with free solution electrokinetic oligonucleotide transfer. Free solution electrokinetics allows coupling of affinity selection and amplification for closed loop oligonucleotide enrichment without the need for offline processes, flow handling components or gel components, while bead based selection and amplification allow efficient manipulation of reagents and reaction products thereby realizing on-chip loop closure and integration of the entire SELEX process. Thus the approach is capable of multi-round enrichment of oligonucleotides using simple transfer processes while maintaining a high level of device integration, as demonstrated by the isolation of an aptamer pool against a protein target (IgA) with significantly higher binding affinity than the starting library in approximately 4 hours of processing time.

An acetyltransferase-independent function of Eso1 regulates centromere cohesion.

Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes.

Functional interplay between cohesin and Smc5/6 complexes.

Chromosomes are subjected to massive reengineering as they are replicated, transcribed, repaired, condensed, and segregated into daughter cells. Among the engineers are three large protein complexes collectively known as the structural maintenance of chromosome (SMC) complexes: cohesin, condensin, and Smc5/6. As their names suggest, cohesin controls sister chromatid cohesion, condensin controls chromosome condensation, and while precise functions for Smc5/6 have remained somewhat elusive, most reports have focused on the control of recombinational DNA repair. Here, we focus on cohesin and Smc5/6 function. It is becoming increasingly clear that the functional repertoires of these complexes are greater than sister chromatid cohesion and recombination. These SMC complexes are emerging as interrelated and cooperating factors that control chromosome dynamics throughout interphase. However, they also release their embrace of sister chromatids to enable their segregation at anaphase, resetting the dynamic cycle of SMC-chromosome interactions.

H2A.Z-dependent regulation of cohesin dynamics on chromosome arms.

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.

Nse1-dependent recruitment of Smc5/6 to lesion-containing loci contributes to the repair defects of mutant complexes.

Of the three structural maintenance of chromosomes (SMC) complexes, Smc5/6 remains the most poorly understood. Genetic studies have shown that Smc5/6 mutants are defective in homologous recombination (HR), and consistent with this, Smc5/6 is enriched at lesions. However, Smc5/6 is essential for viability, but HR is not, and the terminal phenotype of null Smc5/6 mutants is mitotic failure. Here we analyze the function of Nse1, which contains a variant RING domain that is characteristic of ubiquitin ligases. Whereas deletion of this domain causes DNA damage sensitivity and mitotic failure, serine mutations in conserved cysteines do not. However, these mutations suppress the DNA damage sensitivity of Smc5/6 hypomorphs but not that of HR mutants and remarkably decrease the recruitment of Smc5/6 to loci containing lesions marked for HR-mediated repair. Analysis of DNA repair pathways in suppressed double mutants suggests that lesions are channeled into recombination-dependent and error-free postreplication repair. Thus the HR defect in Smc5/6 mutants appears to be due to the presence of dysfunctional complexes at lesions rather than to reflect an absolute requirement for Smc5/6 to complete HR.

Methods for studying the G2 DNA damage checkpoint in mammalian cells.

In response to post-replicative DNA damage, cells activate the G2 DNA damage checkpoint to ensure mitosis is not attempted until the damage has been repaired. This is a common response to a variety of DNA damaging agents, including ionizing radiation and many chemotherapeutic agents used in the treatment of cancer. The G2 DNA damage checkpoint acts to inhibit the mitotic cyclin-dependent kinase, and thus cells are arrested in the G2 phase of the cell cycle. The kinetics of this checkpoint can be assayed by staining cells for markers of mitosis, which can then be quantified by flow cytometry or microscopy.

Methods for studying checkpoint kinases - Chk1.

Attempts to passage through mitosis with unrepaired DNA damage or incompletely replicated DNA leads to genome instability and/or cell death. To prevent this from occurring, an ancient checkpoint (known as the G2 DNA damage checkpoint) that inhibits the activation of the mitotic cyclin-dependent kinase is activated to hold cells in the G2 phase of the cell cycle. The effector of this checkpoint is Chk1, a protein serine-threonine kinase. Chk1 contains an N-terminal catalytic domain, and C-terminal regulatory domain. Within the regulatory domain there are two residues, Serine-317 (S317) and Serine-345 (S345), which are phosphorylated in active Chk1 molecules, and subsequently dephosphorylated to inactivate Chk1 and allow mitotic entry. Phospho-specific antibodies can be used to detect these activating phosphorylations, and this provides a simple and sensitive marker of Chk1 activation.

SMC complexes and topoisomerase II work together so that sister chromatids can work apart.

The pairing of sister chromatids in interphase facilitates error-free homologous recombination (HR). Sister chromatids are held together by cohesin, one of three Structural Maintenance of Chromosomes (SMC) complexes. In mitosis, chromosome condensation is controlled by another SMC complex, condensin, and the type II topoisomerase (Top2). In prophase, cohesin is stripped from chromosome arms, but remains at centromeres until anaphase, whereupon it is removed via proteolytic cleavage. The third SMC complex, Smc5/6, is generally described as a regulator of HR-mediated DNA repair. However, cohesin and condensin are also required for DNA repair, and HR genes are not essential for cell viability, but the SMC complexes are. Smc5/6 null mutants die in mitosis, and in fission yeast, Smc5/6 hypomorphs show lethal mitoses following genotoxic stress, or when combined with a Top2 mutant, top2-191. We found these mitotic defects are due to retention of cohesin on chromosome arms. We also show that Top2 functions in the cohesin cycle, and accumulating data suggests this is not related to its decatenation activity. Thus the SMC complexes and Top2 functionally interact, and any DNA repair function ascribed to Smc5/6 is likely a reflection of a more fundamental role in the regulation of chromosome structure.

Regulation of chk1.

Chk1 is a serine/threonine protein kinase that is the effector of the G2 DNA damage checkpoint. Chk1 homologs have a highly conserved N-terminal kinase domain, and a less conserved C-terminal regulatory domain of ~200 residues. In response to a variety of genomic lesions, a number of proteins collaborate to activate Chk1, which in turn ensures that the mitotic cyclin-dependent kinase Cdc2 remains in an inactive state until DNA repair is completed. Chk1 activation requires the phosphorylation of residues in the C-terminal domain, and this is catalyzed by the ATR protein kinase. How phosphorylation of the C-terminal regulatory domain activates the N-terminal kinase domain has not been elucidated, though some studies have suggested that this phosphorylation relieves an inhibitory intramolecular interaction between the N- and C-termini. However, recent studies in the fission yeast Schizosaccharomyces pombe have revealed that there is more to Chk1 regulation than this auto-inhibition model, and we review these findings and their implication to the biology of this genome integrity determinant.

Brc1-mediated DNA repair and damage tolerance.

The structural maintenance of chromosome (SMC) proteins are key elements in controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes have been defined: cohesin, condensin, and the Smc5/6 complex. The latter is essential for DNA damage responses; in its absence both repair and checkpoint responses fail. In fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74), is suppressed by mild overexpression of a six-BRCT-domain protein, Brc1. Deletion of brc1 does not result in a hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6 complex has remained unclear. Here we show that brc1Delta cells are hypersensitive to a range of radiomimetic drugs that share the feature of creating lesions that are an impediment to the completion of DNA replication. Through a genetic analysis of brc1Delta epistasis and by defining genes required for Brc1 to suppress smc6-74, we find that Brc1 functions to promote recombination through a novel postreplication repair pathway and the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through overproduction of Brc1 bypasses a repair defect in smc6-74, reestablishing resolution of lesions by recombination.