Public Health Research Institute Center
New Jersey Medical School - Rutgers, The State University of New Jersey
225 Warren Street
Newark, New Jersey 07103
Phone: (973) 854-3370
For the past forty-seven years, our laboratory has been exploring nucleic acid structure
to understand the role that it plays in macromolecular interactions that control biological processes. The work has led to the design of novel nucleic acid molecules and the development of experimental techniques that enable the construction of extremely sensitive and specific molecular diagnostic
assays. More than one hundred people have worked in the laboratory or participated as close collaborators. The following paragraphs provide a sketch of some of the significant research themes
that the laboratory has pursued.
Mechanism of RNA replication
The laboratory studied the mechanism of RNA-directed RNA synthesis catalyzed by the bacteriophage polymerase, Qß replicase. No one knew the mechanism by which the viral replicase selectively copies Qß genomic RNA, while ignoring the vast number of other RNA molecules that are present in the bacterial host. Qß RNA was too large to be studied with the techniques that were then available. However, we discovered a much smaller RNA (MDV-1 RNA) in Qß-infected Escherichia coli that is replicated in the same manner as Qß RNA (Kacian et al., 1972). Using classical enzymatic and electrophoretic techniques, we determined the complete nucleotide sequence of both complementary strands of MDV-1 RNA (Mills et al., 1973). This was the longest nucleic acid that had ever been sequenced. Knowledge of the sequence enabled experiments to be carried out that provided insights into the mechanism of RNA replication. We discovered that each complementary strand of MDV-1 RNA possessed extensive secondary structures (Klotz et al., 1980). We demonstrated that the rate of RNA synthesis was determined by pauses in polymerization that occur where secondary structures form in the nascent strand (Mills et al., 1978), and we showed that structural reorganizations occur during product strand elongation (Kramer & Mills, 1981). We also developed an electrophoretic technique for separating the complementary strands that enabled the elucidation of the overall mechanism of RNA-directed RNA synthesis (Dobkin et al., 1979), and we utilized chemical and enzymatic nucleic acid modification methods to identify the sequences and structures that are required for the selective recognition of the RNA by the replicase, and for the initiation of product strand synthesis (Mills et al., 1980; Bausch et al., 1983; Nishihara et al., 1983).
Novel nucleic acid sequencing techniques
Rapid nucleic acid sequence analysis was essential to further studies of replication. We developed a chain-termination method for RNA sequence analysis (Kramer & Mills, 1978) at the same time that Fred Sanger developed a chain-termination method for DNA sequence analysis. Knowledge of the extensive secondary structure of MDV-1 RNA led us to realize that both of these sequencing techniques were compromised by the persistence of strong secondary structures during electrophoretic separation of the partially synthesized strands. We introduced a widely adopted solution to this problem, which was based on the use of modified nucleosides, such as inosine, that form weaker secondary structures (Mills & Kramer, 1979). Years later, we conceived novel techniques that enable entire genomes to be sequenced in a concerted manner by hybridization to oligonucleotide arrays (Chetverin & Kramer, 1993; 1994). These techniques were licensed exclusively to the Affymetrix Corporation (U.S. Patents 6,103,463 and 6,322,971).
In vitro evolution of replicating RNA populations
The in vitro replication of RNA by Qß replicase provides a model system for studying precellular evolution. When MDV-1 RNA is replicated in vitro, the number of RNA molecules doubles every 20 seconds, resulting in an exponential increase in the number of RNA strands. Occasionally, errors occur during replication, producing RNA molecules with a mutated nucleotide sequence. When replication is carried out in the presence of an inhibitor of replication, mutant molecules that resist the inhibitor have a selective advantage, and if allowed to replicate for hundreds of generations, these mutants become predominant in the RNA population. Since phenotype and genotype reside in the same molecule, sequence analysis of the selected RNAs provided insights into the mechanism of Darwinian evolution.
Our laboratory carried out extensive studies on the in vitro evolution of replicating populations of MDV-1 RNA. Utilizing serial transfer techniques, hundreds of replicative generations could be completed in a day. By imposing different selective pressures, different variants emerged. Sequence analysis of the replicating RNA populations at different times during their evolution elucidated how the nucleotide changes that occurred conferred resistance to the particular inhibitor that was used (Kramer et al., 1974). Parallel molecular evolution experiments carried out in the presence of the chain elongation inhibitor, ethidium bromide, confirmed that many different genotypic pathways lead to the same phenotypic result, just as in the evolution of organisms. These experiments laid the foundation for modern in vitro selection techniques that are used to isolate nucleic acid molecules possessing predetermined catalytic activities.
The results of the in vitro evolution experiments also provided useful insights into the structural constraints that are required for an RNA to be replicatable. Though mutations occur everywhere in an RNA, the only mutations selected during the evolution of MDV-1 RNA occurred in single-stranded regions of the molecule, indicating that double-stranded structures are essential to the replicative process. When ribonuclease T1 was used as a selective agent, the mutants that arose were significantly resistant to the nuclease. The macromolecular dimensions of both the nuclease and the RNA limited cleavage to only a few sites on the exterior of the RNA molecule. The selected RNAs possessed non-cleavable nucleotide substitutions at just those exposed sites. These experiments elucidated the tertiary structure of MDV-1 RNA, enabling us to design exponentially amplifiable recombinant RNAs.
Many investigators wished to use the exponential amplification of RNA by Qß replicase to synthesize large amounts of any mRNA or any genomic RNA. However, Qß replicase is highly specific for Qß phage RNA. We devised a scheme that enabled the replication of any heterologous RNA. Novel RNA templates were constructed by covalently inserting heterologous RNA sequences within the MDV-1 sequence at a single-stranded site that occurs on the exterior of the MDV-1 RNA molecule (Miele et al., 1983). The resulting recombinant RNAs possessed all of the secondary and tertiary structures that are required for replication, and the presence of the inserted sequence on the exterior of the molecule did not interfere with access to the structures required for replication. Consequently, Qß replicase was able to catalyze the exponential synthesis of the entire recombinant RNA. Moreover, the recombinant RNAs were bifunctional, in that they retained the biological activity of the inserted sequence, as well as the replicatability of the MDV-1 RNA.
We constructed recombinant RNAs that contained the entire mRNA sequence encoding chloramphenicol acetyltransferase. These recombinant molecules were amplified exponentially in vitro by incubation with Qß replicase, and the replicated RNA served as template for the cell-free synthesis of enzymatically active chloramphenicol acetyltransferase (Wu et al., 1992). We demonstrated that these recombinant mRNAs could be continuously synthesized and that large quantities of biologically active protein could be produced in a coupled replication-translation system that contained both Qß replicase and bacterial ribosomes (Ryabova et al., 1994). We also constructed amplifiable recombinant RNAs that contained entire viroid genomes (U.S. Patent 5,871,976), and the recombinant, by itself, was infectious when placed on the leaves of tomato plants.
Extremely sensitive gene detection assays
With the advent of the AIDS crisis, it became imperative that very sensitive assays be developed for the detection of pathogenic retroviruses. We realized that an attractive strategy for detecting rare targets is to link a nucleic acid probe to a replicatable reporter that can be amplified exponentially after hybridization to reveal the presence of the target (Chu et al., 1986). We therefore covalently linked MDV-1 RNA to an oligonucleotide probe that was complementary to a predetermined genetic target. The resulting molecules were used in assays in which the probes bind specifically to target sequences, unbound probes are washed away, and the probe-target hybrids are incubated with Qß replicase to generate a large number of easily detected reporter molecules. Since as little as a single molecule of MDV-1 RNA can serve as template for the exponential synthesis of millions of RNA copies by Qß replicase, these assays were extremely sensitive.
We also realized that it was simpler to perform these assays with recombinant MDV-1 RNA molecules in which a probe sequence is embedded within the MDV-1 RNA, rather than being attached to the RNA by a linker. We constructed recombinant-RNA probes and demonstrated that they were bifunctional, in that they bound specifically to their targets, and after they were bound they served as templates for their own exponential amplification (Lizardi et al., 1988). We demonstrated that recombinant-RNA hybridization probes could be used in sensitive gene detection assays (Lomeli et al., 1989; Kramer et al., 1992). The inclusion of intercalating fluorescent dyes, such as ethidium bromide, in the reaction mixtures to detect the reporter RNA enabled the assays to be carried out in real-time under homogeneous conditions in sealed tubes (Kramer & Lizardi, 1989; Lomeli et al., 1989). We also demonstrated that the time required to synthesize a given quantity of reporter RNA is inversely proportional to the logarithm of the number of target molecules originally present in a sample, thus enabling quantitative determinations over an extremely wide range of target concentrations (U.S. Patent 5,503,979). This quantitative analytical technique has found wide application in real-time clinical assays that utilize polymerase chain reactions.
The sensitivity of Qß replicase assays employing recombinant RNAs was limited by the inability to wash away every unbound probe. Persistent nonhybridized probes were amplified along with hybridized probes, generating a background signal that obscured the presence of rare targets. We investigated a number of different ways to eliminate this background (Kramer & Lizardi, 1989; Blok et al., 1997; U.S. Patents 5,118,801 and 5,312,728). Rather than trying to improve existing washing techniques (which were already quite efficient), we altered the design of the probes so that they could not be replicated unless they were hybridized to their target. We divided the recombinant-RNA probes into two separate molecules, neither of which could be amplified by itself, because neither contained all of the elements of sequence and structure that are required for replication by Qß replicase. The division site was located in the middle of the embedded probe sequence. When these "binary probes" were hybridized to adjacent positions on their target sequence, they could be joined to each other by incubation with an appropriate ligase, generating a replicatable reporter RNA, which was then exponentially amplified by incubation with Qß replicase. Nonhybridized probes, on the other hand, because they were not aligned on a target, could not be ligated, and signal generation was strictly dependent on the presence of target molecules. Because there were no background signals, the resulting assays were extraordinarily sensitive. As little as a single HIV-1 infected cell could be detected in samples containing 100,000 uninfected lymphocytes (Tyagi et al., 1996). This technique was licensed to Abbott Laboratories (U.S. Patents 5,759,773 and 5,807,674) and has been used in automated assays that detect the genes of many different infectious agents in human clinical samples.
We invented novel hybridization probes called "molecular beacons," which enable the direct detection of specific nucleic acids in living cells and in diagnostic assays (Tyagi & Kramer, 1996). These probes are hairpin-shaped oligonucleotides with a fluorophore at one end and a nonfluorescent quencher at the other end. When they are not bound to a target nucleic acid, the fluorophore is in contact with the quencher and the probes are dark. When these probes bind to their targets, they undergo a conformational reorganization that separates the fluorophore from the quencher, resulting in a bright fluorescent signal that indicates the presence of the target. Because these probes only fluoresce when they are bound to target sequences, there is no need to isolate the probe-target hybrids to determine the amount of target present in a sample.
We showed that the mechanism of fluorescence quenching involves the transient formation of a nonfluorescent fluorophore-quencher complex, thus any desired fluorophore can be used as a label (Tyagi et al., 1998; Marras et al., 2002). When a set of molecular beacons are prepared, each specific for a different target sequence, and each labeled with a differently colored fluorophore, different nucleic acid targets can be detected simultaneously in the same assay tube or in the same cell. Moreover, by taking their thermodynamic behavior into consideration (Bonnet et al., 1999), molecular beacons can be designed so that they are significantly more specific than corresponding conventional linear hybridization probes. Molecular beacons can be designed in such a manner that the presence of even a single nucleotide substitution in a target sequence prevents the formation of a probe-target hybrid (Tyagi et al., 1998; Marras et al., 1999).
Our laboratory demonstrated the advantages of using molecular beacons as amplicon detector probes in quantitative, real-time, exponential amplification assays. We designed extremely sensitive, multiplex, clinical PCR assays that simultaneously detect four different pathogenic retroviruses in blood (Vet et al., 1999); and we designed "wavelength-shifting" molecular beacons (Tyagi et al., 2000) that enable many different genetic targets to be detected simultaneously in the same sample, utilizing simple instruments that possess a monochromatic light source. We also pioneered the use of molecular beacons for high-throughput "spectral genotyping" (Kostrikis et al., 1998); and we demonstrated the ease with which molecular beacons can distinguish single-nucleotide polymorphisms in PCR assays (Marras et al., 1999). We showed that molecular beacons work well in NASBA assays (Van Beuningen et al., 2001), as well as in PCR assays; and we demonstrated how molecular beacons can be used to monitor in vitro transcription in real time (Marras et al., 2004).
Our laboratory also designed a panel of assays that identify mutations in potential parents that cause Tay-Sachs disease and cystic fibrosis in the children of Ashkenazi Jews; and we developed a single-tube, multiplex assay that utilizes molecular beacons for the detection of bacteria that can be used as agents of bioterror: Yersinia pestis, Bacillus anthracis, Burkholderia mallei, and Francisella tularensis. We also developed a single-tube version of a PCR assay that rapidly identifies multidrug-resistant Mycobacterium tuberculosis in sputum samples (El-Hajj et al., 2001). This assay underwent clinical trials (Varma-Basil et al., 2004), was developed for commercial distribution, was endorsed by the World Health Organization, and is now the principal assay for the direct detection of tuberculosis utilized throughout the world. We have also contributed to the development of assays that detect hospital-acquired infections caused by pathogenic fungi and by methicillin-resistant and vancomycin-resistant Staphylococcus aureus.
Highly multiplex screening assays
Our laboratory has developed multiplex screening assays that utilize color-coded molecular beacons in single-tube gene amplification reactions that identify which infectious agent, if any, is present in a clinical sample (U.S. Patents 7,385,043 and 7,771,949). The first assay of this type is able to identify the 15 most prevalent bacterial species that are found in blood samples taken from febrile patients (Marras et al., 2017). Unlike classical blood cultures, which take many days to yield results, these “molecular blood cultures” require only two hours to complete. Each of the 15 species-specific molecular beacons is labeled with a unique combination of two differently colored fluorophores selected from a palette of six differently colored fluorophores. The two-color fluorescence signal that arises during the course of a PCR assay that amplifies a segment of the bacterial 16S ribosomal RNA gene uniquely identifies the species that is present. Future assays will utilize three differently colored fluorophores (selected from a palette of seven colors) to uniquely label each of 35 species-specific molecular beacons. This will enable simultaneous screening for the presence of both common species and rarely seen species, such as agents of bioterror. Widespread use of these assays will enable the rapid identification of common infectious agents, while at the same time providing an early warning system that will help contain the spread of major epidemics.
We have also designed highly multiplex screening assays based on a different principle. In these assays, only four differently colored molecular beacons are present during the amplification of a segment of the bacterial 16S ribosomal RNA gene. Unlike the assays described above, these molecular beacons contain relatively long probe sequences, enabling them to bind to amplified 16S ribosomal RNA gene segments generated from many different bacterial species. The stability of each of the four resulting probe-target hybrids depends upon how well each of the molecular beacons matches the amplified target sequence. After amplification, the mixture of fluorescent probe-target hybrids is melted apart
by raising the temperature and simultaneously determining, for each of the four differently colored probes, the temperature at which each hybrid falls apart (seen as a loss of fluorescence). The resulting set of four melting temperatures serves as a unique spectral signature that identifies which species is present (U.S. Patents 7,662,550 and 9,260,761). We demonstrated that 27 different species of mycobacteria can be uniquely identified with the aid of only four of these “sloppy” molecular beacons (El-Hajj et al., 2009); and we demonstrated that sloppy molecular beacon probes can identify 94 different species of bacteria (across 64 genera) in rapid, PCR assays designed to detect and identify bacterial species that can cause sepsis if present in the blood stream (Chakravorty et al., 2010).
Self-reporting oligonucleotide arrays
We have demonstrated that molecular beacons are useful for the determination of gene expression profiles (Manganelli et al., 1999; Dracheva et al., 2001). We are exploring the use of arrays of molecular beacons for the simultaneous quantitation of hundreds of different mRNAs in a sample. Each molecular beacon is immobilized at a different location on the surface of a glass chip. Instead of enzymatically adding a fluorophore to the target mRNAs and hybridizing those targets to an array of linear probes, when an array consists of immobilized molecular beacons, the mRNAs need not be labeled and the molecular beacons become fluorescent when the targets bind to them. Hairpin-shaped probes are significantly more specific than linear probes, and the intensity of the fluorescence generated by the molecular beacons is directly proportional to the number of mRNAs that are bound.
We are also investigating distributed array formats, in which many different molecular beacon probes are used at the same time. Each type of molecular beacon probe is immobilized on a different microbead, and tens of thousands of beads are used in an assay. After hybridization to a mixture of mRNAs, the fluorescence of each bead is rapidly read by a spectral analyzer that determines the number of target mRNAs bound to each bead from the fluorescence of the molecular beacons on its surface. In order to facilitate this approach, we have developed a rapid method for telling which bead contains which probe (U.S. Patent 7,741,031). In this technique, additional hairpin-shaped nucleic acids possessing quenchers and differently colored fluorophores at each end are also immobilized on the surface of each bead. These additional hairpins do not serve as probes; instead the presence or absence of each hairpin serves as a binary element in a “serial number” that identifies the bead to which they are attached. For example, three different-length hairpins can be used, each labeled with one of five differently colored fluorophores, for a total of 15 distinctive elements that can be present or absent on the surface of the bead. The serial number of each bead in a collection of perhaps 100,000 beads is then simultaneously read by raising the temperature and noting, for each bead, which fluorescent colors appear on the surface of the bead as the temperature is raised, causing the three different-length hairpins to denature. The availability of practical gene expression profiling arrays should enable the identification of gene ensembles that control development, the discovery of new metabolic pathways, the exploration of cellular responses to viral and bacterial infection, and the development of high-throughput assays that identify new therapeutic agents.
Detection of mRNAs in living cells
One of the most exciting programs in the laboratory is the direct detection of mRNAs in living cells. Conventional in situ hybridization techniques require the "fixing" of cells to enable the unbound probes to be washed away. Fixing denatures and crosslinks the proteins, resulting in cell death. Thus, in situ hybridization provides a static view of mRNA distribution and is not effective for the investigation of dynamic processes. Because molecular beacons only become fluorescent when they bind to their target, there is no need to fix and wash the cells, and the synthesis, movement, localization, and disappearance of mRNAs can be viewed as a function of time. We have shown that molecular beacons are excellent probes for visualizing mRNAs in living cells, and we have used them in experiments with many different cell types. We found that molecular beacons can be synthesized from modified nucleotides that do not occur naturally, such as the 2'-O-methylribonucleotides, in order to prevent digestion of the molecular beacons by cellular nucleases and to prevent cleavage of the target mRNAs by cellular ribonuclease H. We also found that the interfering effects of autofluorescence from cellular components can be overcome by using wavelength-shifting molecular beacons, which have large Stokes shifts that enable them to fluoresce at longer wavelengths (Tyagi et al., 2000). Furthermore, molecular beacons are not toxic to cells, and different mRNAs in the same cell can be visualized simultaneously with differently colored molecular beacons. And finally, we have linked molecular beacons to tRNA sequences in order to ensure that the probes are retained within the cytoplasm (Mhlanga et al., 2005).
The injection of molecular beacons into living cells allows the expression of particular genes to be monitored as a function of genetically programmed development, or as a response to external stimulation. With the aid of deconvolving and confocal fluorescence microscopy, we used molecular beacons to visualize the formation, transport, and localization of oskar mRNA in living Drosophila embryos (Bratu et al., 2003). We also used molecular beacons to follow the movement of b-actin mRNA into growing lamellipodia as lymphocytes move across surfaces. Currently, we are using molecular beacons to track the movement and localization of CaMKII, Map-2, b-actin, and Arc mRNA
in primary cultures of rat hippocampal neurons, in order to understand how the stimulation of presynaptic dendrites leads to mRNA localization and to the long-term potentiation of postsynaptic dendrites, which is an attractive model system for studying cellular mechanisms of memory formation (Batish et al., 2011). In addition, we are following the transport of specific mRNAs from the neuronal nucleus to postsynaptic dendritic sites, to determine the kinetics of mRNA movement and to elucidate
the mechanism by which mRNAs are localized in stimulated dendrites.
Tracking Individual mRNA molecules
Although the fluorescence from a single molecular beacon bound to an mRNA is not sufficiently bright to be seen above the background fluorescence in a living cell, we devised a method that enables 96 molecular beacons to bind to a single mRNA molecule, which allows specific mRNAs to be seen and followed as they are synthesized, processed, and move within the nucleus and through the nuclear pores to the cytoplasm (Vargas et al., 2005). The technique that we developed involves the cloning of a synthetic sequence into the region of a target gene that encodes the 3'-untranslated region of the particular mRNA molecules that we wish to see and follow. The synthetic sequence contains 96 tandemly repeated molecular beacon binding sites. The presence of 96 probes on the 3' end of each mRNA does not prevent the binding of nuclear proteins. The motion of these individual mRNA-protein complexes were recorded by time-lapse photography. Analysis of their tracks demonstrates that they move freely by Brownian diffusion within the extranucleolar, interchromatin space. Experimental manipulation of the cellular environment by lowering the temperature and altering the availability of ATP, enabled us to conclude that occasionally these particles become trapped on the surface of the chromatin, and that the expenditure of metabolic energy is required for the particles to resume their motion. We are now introducing tandemly repeated molecular beacon target sites into different genes in order to study the mechanism of transport and localization of particular mRNAs in different cell types. This method will also aid in the identification of cellular sites where other processes central to gene expression occur. Examples of such processes are mRNA splicing, maturation, export, and decay. The ability to simultaneously track different mRNAs tagged with different multimeric target sequences, using differently colored molecular beacons in the same cell, will be especially useful in this regard.
Detection of somatic mutations that characterize cancer cells
Our laboratory has long been interested in developing PCR assays that use allele-discriminating gene amplification primers to detect DNA containing rare somatic mutations that are characteristic of cancer cells, without interference from far more abundant related wild-type DNA (U.S. Patents 6,277,607 and 6,365,729). Recently, we designed “SuperSelective” PCR primers that are able to detect as few as 10 mutant DNA fragments in the presence of 1,000,000 related wild-type fragments (Patent Publication WO2014124290 A1). Moreover, we recently added additional design elements to these primers that enable multiplex PCR assays to be carried out. The goal of this work is to develop an inexpensive assay that can be carried out with a blood sample obtained during a routine annual medical checkup. The assay would be so sensitive that it could detect cancer anywhere in a person’s body before any symptoms have occurred. Early detection of actionable mutations is likely to improve treatment and may possibly lead to cures. Moreover, since only a routine blood sample is taken, patients with cancer can be followed periodically, and treatment can be adjusted in accordance with individual results.
Vargas DY, Kramer FR, Tyagi S, Marras SAE (2016) Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS One 11: e0156546. PMI: 27244445
Tyagi S, Kramer FR (2012) Molecular Beacons in Diagnostics. Faculty of 1000 Medicine Reports 4: 10.
Batish M, van den Bogaard P, Kramer FR, Tyagi S (2012) Neuronal mRNAs travel singly into dendrites. Proc Natl Acad Sci U S A. PMI: 22392993
Altan-Bonnet G, Kramer FR (2012) Nucleic acid hybridization: Robust sequence discrimination. Nat Chem 4: 155-157. PMI: 22354426
Chakravorty S, Aladegbami B, Burday M, Levi M, Marras SA, Shah D, El-Hajj HH, Kramer FR, Alland D (2010) Rapid universal identification of bacterial pathogens from clinical cultures by using a novel sloppy molecular beacon melting temperature signature technique. J Clin Microbiol 48: 258-267. PMI: 19923485
El-Hajj HH, Marras SA, Tyagi S, Shashkina E, Kamboj M, Kiehn TE, Glickman MS, Kramer FR, Alland D (2009) Use of sloppy molecular beacon probes for identification of mycobacterial species. J Clin Microbiol 47: 1190-1198. PMI: 19171684
Marras SA, Tyagi S, Kramer FR (2006) Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes. Clin Chim Acta 363: 48-60. PMI: 16111667
Vargas DY, Raj A, Marras SA, Kramer FR, Tyagi S (2005) Mechanism of mRNA transport in the nucleus. Proc Natl Acad Sci U S A 102: 17008-17013. PMI: 16284251
Mhlanga MM, Vargas DY, Fung CW, Kramer FR, Tyagi S (2005) tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells. Nucleic Acids Res 33: 1902-1912. PMI: 15809226
Varma-Basil M, El-Hajj H, Marras SA, Hazbon MH, Mann JM, Connell ND, Kramer FR, Alland D (2004) Molecular beacons for multiplex detection of four bacterial bioterrorism agents. Clin Chem 50: 1060-1062. PMI: 15161722
Varma-Basil M, El-Hajj H, Colangeli R, Hazbon MH, Kumar S, Bose M, Bobadilla-del-Valle M, Garcia LG, Hernandez A, Kramer FR, Osornio JS, Ponce-de-Leon A, Alland D (2004) Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay. J Clin Microbiol 42: 5512-5516. PMI: 15583274
Marras SA, Gold B, Kramer FR, Smith I, Tyagi S (2004) Real-time measurement of in vitro transcription. Nucleic Acids Res 32: e72. PMI: 15155820
Marras SA, Kramer FR, Tyagi S (2003) Genotyping SNPs with molecular beacons. Methods Mol Biol 212: 111-128. PMI: 12491906
Bratu DP, Cha BJ, Mhlanga MM, Kramer FR, Tyagi S (2003) Visualizing the distribution and transport of mRNAs in living cells. Proc Natl Acad Sci U S A 100: 13308-13313. PMI: 14583593
Marras SA, Kramer FR, Tyagi S (2002) Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res 30: e122. PMI: 12409481
Van Beuningen R, Marras SAE, Kramer FR, Oosterlaken T, Weusten J, Borst G, Van de Wiel P (2001) Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons. Proceedings - SPIE the International Society for Optical Engineering 4264: 66-71.
El-Hajj HH, Marras SA, Tyagi S, Kramer FR, Alland D (2001) Detection of rifampin resistance in Mycobacterium tuberculosis in a single tube with molecular beacons. J Clin Microbiol 39: 4131-4137. PMI: 11682541
Dracheva S, Marras SA, Elhakem SL, Kramer FR, Davis KL, Haroutunian V (2001) N-methyl-D-aspartic acid receptor expression in the dorsolateral prefrontal cortex of elderly patients with schizophrenia. Am J Psychiatry 158: 1400-1410. PMI: 11532724
Xiao G, Chicas A, Olivier M, Taya Y, Tyagi S, Kramer FR, Bargonetti J (2000) A DNA damage signal is required for p53 to activate gadd45. Cancer Res 60: 1711-1719. PMI: 10749144
Tyagi S, Marras SA, Kramer FR (2000) Wavelength-shifting molecular beacons. Nat Biotechnol 18: 1191-1196. PMI: 11062440
Piatek AS, Telenti A, Murray MR, El-Hajj H, Jacobs WR, Jr., Kramer FR, Alland D (2000) Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing. Antimicrob Agents Chemother 44: 103-110. PMI: 10602730
Vet JA, Majithia AR, Marras SA, Tyagi S, Dube S, Poiesz BJ, Kramer FR (1999) Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci U S A 96: 6394-6399. PMI: 10339598
Rhee JT, Piatek AS, Small PM, Harris LM, Chaparro SV, Kramer FR, Alland D (1999) Molecular epidemiologic evaluation of transmissibility and virulence of Mycobacterium tuberculosis. J Clin Microbiol 37: 1764-1770. PMI: 10325321
Marras SA, Kramer FR, Tyagi S (1999) Multiplex detection of single-nucleotide variations using molecular beacons. Genet Anal 14: 151-156. PMI: 10084107
Manganelli R, Dubnau E, Tyagi S, Kramer FR, Smith I (1999) Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis. Mol Microbiol 31: 715-724. PMI: 10027986
Cayouette M, Sucharczuk A, Moores J, Tyagi S, Kramer FR (1999) Using molecular beacons to monitor PCR product formation. Strategies 12: 85-92.
Bonnet G, Tyagi S, Libchaber A, Kramer FR (1999) Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci U S A 96: 6171-6176. PMI: 10339560
Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16: 49-53. PMI: 9447593
Piatek AS, Tyagi S, Pol AC, Telenti A, Miller LP, Kramer FR, Alland D (1998) Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat Biotechnol 16: 359-363. PMI: 9555727
Leone G, van Schijndel H, van Gemen B, Kramer FR, Schoen CD (1998) Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res 26: 2150-2155. PMI: 9547273
Kostrikis LG, Tyagi S, Mhlanga MM, Ho DD, Kramer FR (1998) Spectral genotyping of human alleles. Science 279: 1228-1229. PMI: 9508692
Gao W, Tyagi S, Kramer FR, Goldman E (1997) Messenger RNA release from ribosomes during 5'-translational blockage by consecutive low-usage arginine but not leucine codons in Escherichia coli. Mol Microbiol 25: 707-716. PMI: 9379900
Blok HJ, Kramer FR (1997) Amplifiable hybridization probes containing a molecular switch. Mol Cell Probes 11: 187-194. PMI: 9232617
Tyagi S, Landegren U, Tazi M, Lizardi PM, Kramer FR (1996) Extremely sensitive, background-free gene detection using binary probes and beta replicase. Proc Natl Acad Sci U S A 93: 5395-5400. PMI: 8643586
Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14: 303-308. PMI: 9630890
Hsuih TC, Park YN, Zaretsky C, Wu F, Tyagi S, Kramer FR, Sperling R, Zhang DY (1996) Novel, ligation-dependent PCR assay for detection of hepatitis C in serum. J Clin Microbiol 34: 501-507. PMI: 8904402
Ryabova L, Volianik E, Kurnasov O, Spirin A, Wu Y, Kramer FR (1994) Coupled replication-translation of amplifiable messenger RNA. A cell-free protein synthesis system that mimics viral infection. J Biol Chem 269: 1501-1505. PMI: 8288616
Chetverin AB, Kramer FR (1994) Oligonucleotide arrays: new concepts and possibilities. Biotechnology (N Y) 12: 1093-1099. PMI: 7765552
Chetverin AB, Kramer FR (1993) Sequencing of pools of nucleic acids on oligonucleotide arrays. Biosystems 30: 215-231. PMI: 8374077
Wu Y, Zhang DY, Kramer FR (1992) Amplifiable messenger RNA. Proc Natl Acad Sci U S A 89: 11769-11773. PMI: 1465396
Lizardi PM, Kramer FR (1991) Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases. Trends Biotechnol 9: 53-58. PMI: 1366952
Kramer FR, Lizardi PM (1990) Amplifiable hybridization probes. Ann Biol Clin (Paris) 48: 409-411. PMI: 2221501
Lomeli H, Tyagi S, Pritchard CG, Lizardi PM, Kramer FR (1989) Quantitative assays based on the use of replicatable hybridization probes. Clin Chem 35: 1826-1831. PMI: 2673578
Kramer FR, Lizardi PM (1989) Replicatable RNA reporters. Nature 339: 401-402. PMI: 2725660
Lizardi PM, Guerra CE, Lomeli H, Tussie-Luna I, Kramer FR (1988) Exponential amplification of recombinant RNA hybridization probes. Biotechnology 6: 1197-1202.
Priano C, Kramer FR, Mills DR (1987) Evolution of the RNA coliphages: the role of secondary structures during RNA replication. Cold Spring Harb Symp Quant Biol 52: 321-330. PMI: 3331342
Chu BC, Kramer FR, Orgel LE (1986) Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res 14: 5591-5603. PMI: 2426657
LaFlamme SE, Kramer FR, Mills DR (1985) Comparison of pausing during transcription and replication. Nucleic Acids Res 13: 8425-8440. PMI: 3841202
Axelrod VD, Kramer FR (1985) Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators. Biochemistry 24: 5716-5723. PMI: 3002422
Nishihara T, Mills DR, Kramer FR (1983) Localization of the Q beta replicase recognition site in MDV-1 RNA. J Biochem 93: 669-674. PMI: 6192124
Miele EA, Mills DR, Kramer FR (1983) Autocatalytic replication of a recombinant RNA. J Mol Biol 171: 281-295. PMI: 6655695
Bausch JN, Kramer FR, Miele EA, Dobkin C, Mills DR (1983) Terminal adenylation in the synthesis of RNA by Q beta replicase. J Biol Chem 258: 1978-1984. PMI: 6185491
Kramer FR, Mills DR (1981) Secondary structure formation during RNA synthesis. Nucleic Acids Res 9: 5109-5124. PMI: 6171773
Mills DR, Kramer FR, Dobkin C, Nishihara T, Cole PE (1980) Modification of cytidines in a Q beta replicase template: analysis of conformation and localization of lethal nucleotide substitutions. Biochemistry 19: 228-236. PMI: 6986163
Klotz G, Kramer FR, Kleinschmidt AK (1980) Conformational details of partially base-paired small RNAs in the nanometer range. Electron Microscopy 2: 530-531.
Mills DR, Kramer FR (1979) Structure-independent nucleotide sequence analysis. Proc Natl Acad Sci U S A 76: 2232-2235. PMI: 287063
Dobkin C, Mills DR, Kramer FR, Spiegelman S (1979) RNA replication: required intermediates and the dissociation of template, product, and Q beta replicase. Biochemistry 18: 2038-2044. PMI: 107965
Mills DR, Dobkin C, Kramer FR (1978) Template-determined, variable rate of RNA chain elongation. Cell 15: 541-550. PMI: 719752
Kramer FR, Mills DR (1978) RNA sequencing with radioactive chain-terminating ribonucleotides. Proc Natl Acad Sci U S A 75: 5334-5338. PMI: 281683
Mills DR, Kramer FR, Dobkin C, Nishihara T, Speigelman S (1975) Nucleotide sequence of microvariant RNA: another small replicating molecule. Proc Natl Acad Sci U S A 72: 4252-4256. PMI: 1060104
Kramer FR, Mills DR, Cole PE, Nishihara T, Spiegelman S (1974) Evolution in vitro: sequence and phenotype of a mutant RNA resistant to ethidium bromide. J Mol Biol 89: 719-736. PMI: 4449129
Mills DR, Kramer FR, Spiegelman S (1973) Complete nucleotide sequence of a replicating RNA molecule. Science 180: 916-927. PMI: 4706684
Kacian DL, Mills DR, Kramer FR, Spiegelman S (1972) A replicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication. Proc Natl Acad Sci U S A 69: 3038-3042. PMI: 4507621
Kramer FR (1969) Factors affecting translation of messenger RNAs in vitro: use of a GTP analog to investigate rates of polypeptide chain elongation. Doctoral dissertation. The Rockefeller University. Thesis advisor: Vincent Allfrey.: PMI:
Davidson EH, Crippa M, Kramer FR, Mirsky AE (1966) Genomic function during the lampbrush chromosome stage of amphibian oogenesis. Proc Natl Acad Sci U S A 56: 856-863. PMI: 16578646
Kramer FR (1964) The kinetics of deoxyribonuclease action on the lampbrush chromosomes of Triturus. Undergraduate honors thesis. University of Michigan. Thesis advisors: Berwind P. Kaufmann and Helen Gay.: PMI:
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