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`Personal
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`1962 - 1964
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`1969 - 1986
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`1986 - present
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`Education
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`1956 - 1959
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`1959 - 1964
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`1964 - 1969
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`1969 - 1972
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`Appointments
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`Awards
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`1987 - 2014
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`2003 - present
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`Fred Russell Kramer
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`November 9, 2017
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`July 7, 1942 – New York City
`Married forty years, widowed, two children, four grandchildren
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`The Bronx High School of Science
`University of Michigan – B.S. with Honors in Zoology
`The Rockefeller University – Ph.D. (with Vincent Allfrey)
`Columbia University – Postdoctoral training (with Sol Spiegelman)
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`Laboratory Technician, Cytogenetics Laboratory
`Carnegie Institution of Washington, Ann Arbor, Michigan
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`Department of Genetics and Development
` and Institute of Cancer Research
`College of Physicians and Surgeons
`Columbia University
`1969 - 1971
`Fellow of the American Cancer Society
`1971 - 1972
`Research Associate
`1972 - 1973
`Instructor
`1973 - 1980
`Assistant Professor
`1980 - 1983
`Senior Research Associate
`1983 - 1986
`Research Scientist
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`The Public Health Research Institute
`1986 - present
`Co-Director, Laboratory of Molecular Genetics
`2000 - 2006
`Director, PHRI Office of Technology Transfer
`2006 - 2010
`Associate Director of PHRI for Technology Transfer
`2012 - 2017
`Associate Director of PHRI for Business Development
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`Department of Microbiology
`New York University School of Medicine
`1987 - 2003
`Research Professor
`2003 - 2014
`Adjunct Professor
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`Professor of Microbiology, Biochemistry and Molecular Genetics
`Public Health Research Institute, New Jersey Medical School
`2003 - 2013
` University of Medicine and Dentistry of New Jersey
`2013 - present Rutgers, The State University of New Jersey
`2015 - present
`Associate Member, Cancer Institute of New Jersey
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`2005 Jacob Heskel Gabbay Award in Biotechnology and Medicine
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`American Association of University Professors
`American Society for Biochemistry and Molecular Biology
`American Society for Microbiology
`Association for Molecular Pathology
`New York Academy of Sciences
`Society of the Sigma Xi
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`The Johns Hopkins University Exhibit JHU2002 - Page 1 of 20
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`2
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`Fred Russell Kramer
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`Laboratory of Molecular Genetics
`Public Health Research Institute
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`RESEARCH SYNOPSIS
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`For the past forty-eight years, our laboratory has been exploring nucleic acid structure
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`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.
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`Mechanism of RNA replication
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`The laboratory studied the mechanism of RNA-directed RNA synthesis catalyzed by the
`bacteriophage polymerase, Qβ replicase. No one knew the mechanism by which the viral replicase
`selectively copies Qβ genomic RNA, while ignoring the vast number of other RNA molecules that are
`present in the bacterial host. Qβ 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 Qβ-infected Escherichia coli
`that is replicated in the same manner as Qβ 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).
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`Novel nucleic acid sequencing techniques
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`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).
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`In vitro evolution of replicating RNA populations
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`The in vitro replication of RNA by Qβ 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.
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`3
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`Our laboratory carried out extensive studies on the in vitro evolution of replicating populations of
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`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.
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`The results of the in vitro evolution experiments also provided useful insights into the structural
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`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.
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`Recombinant RNAs
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`Many investigators wished to use the exponential amplification of RNA by Qβ replicase to
`synthesize large amounts of any mRNA or any genomic RNA. However, Qβ replicase is highly specific
`for Qβ 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,
`Qβ 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.
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`We constructed recombinant RNAs that contained the entire mRNA sequence encoding
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`chloramphenicol acetyltransferase. These recombinant molecules were amplified exponentially in vitro
`by incubation with Qβ 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 Qβ
`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.
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`Extremely sensitive gene detection assays
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`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 Qβ 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 Qβ
`replicase, these assays were extremely sensitive.
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`We also realized that it was simpler to perform these assays with recombinant MDV-1 RNA
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`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.
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`The sensitivity of Qβ replicase assays employing recombinant RNAs was limited by the inability
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`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 Qβ 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 Qβ 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.
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`Molecular beacons
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`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.
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`We showed that the mechanism of fluorescence quenching involves the transient formation of
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`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).
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`5
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`Our laboratory demonstrated the advantages of using molecular beacons as amplicon detector
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`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).
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`Our laboratory also designed a panel of assays that identify mutations in potential parents that
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`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.
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`Highly multiplex screening assays
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`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.
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`We have also designed highly multiplex screening assays based on a different principle. In these
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`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
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`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).
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`Self-reporting oligonucleotide arrays
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`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.
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`We are also investigating distributed array formats, in which many different molecular beacon
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`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.
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`Detection of mRNAs in living cells
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`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
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`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).
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`7
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`The injection of molecular beacons into living cells allows the expression of particular genes
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`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 β-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, β-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.
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`Tracking Individual mRNA molecules
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`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.
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`Detection of somatic mutations that characterize cancer cells
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`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
`WO/2014/124290). Moreover, we have 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.
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`Bibliography
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`Structure and function of lampbrush chromosomes
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` 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.
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` Davidson EH, Crippa M, Kramer FR, and Mirsky AE (1966) Genomic function during
`the lampbrush chromosome stage of amphibian oogenesis. Proc Natl Acad Sci USA
`56, 856-863.
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`Translation of messenger RNA
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` 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.
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`Sequence and structure of replicating RNAs
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` Kacian DL, Mills DR, Kramer FR, and Spiegelman S (1972) A replicating RNA molecule
`suitable for a detailed analysis of extracellular evolution and replication. Proc Natl Acad
`S