FIELD OF INVENTION

The present invention pertains to the field of nanoscale devices. The present invention also pertains to the field of macromolecular sequencing, particularly the field of DNA sequencing and characterization.

BACKGROUND OF THE INVENTION

Various scientific and patent publications are referred to herein. Each is incorporated by reference in its entirety.

Biomolecules such as DNA or RNA are long molecules composed of nucleotides, the sequence of which is directly related to the genomic and post-genomic gene expression information of an organism. In most cases, the mutation or rearrangement of the nucleotide sequences during an individual's life span can lead to disease states such as genetic abnormalities or cell malignancy. In other cases, the small amount of sequence differences among each individual reflects the diversity of the genetic makeup of the population. Because of these differences in genetic sequence, certain individuals respond differently to environmental stimuli and signals, including drug treatments. For example, some patients experience positive response to certain compounds while others experience no effects or even adverse side effects.

The fields of population genomics, medical genomics and pharmacogenomics studying genetic diversity and medical pharmacological implications require extensive sequencing coverage and large sample numbers. The sequencing knowledge generated would be especially valuable for the health care and pharmaceutical industry. Cancer genomics and diagnostics study genomic instability events leading to tumorigenesis. All these fields would benefit from technologies enabling fast determination of the linear sequence of biopolymer molecules such as nucleic acids, or epigenetic biomarkers such as methylation patterns along the biopolymers. There is a long felt need to use very little amount of sample, even as little as a single cell. This would greatly advance the ability to monitor the cellular state and understand the genesis and progress of diseases such as the malignant stage of a cancer cell.

Most genome or epigenome analysis technologies remain too expensive for general analysis of large genomic regions for a large population. In order to achieve the goal of reducing the genomic analysis cost by at least four orders of magnitude, the so-called “$1000 genome” milestone, new technologies for molecular analysis methods are needed. See “The Quest for the $1,000 Human Genome,” by Nicholas Wade, The New York Times, Jul. 18, 2006.

One technology developed for fast sequencing involves the use of a nanoscale pore through which DNA is threaded. Historically, the “nanopore” concept used a biological molecular device to produce ionic current signatures when RNA and DNA strands are driven through the pore by an applied voltage. Biological systems, however, are sensitive to pH, temperature and electric fields. Further, biological molecules are not readily integrated with the semiconductor processes required for sensitive on-chip electronics.

Many efforts have been since focused on designing and fabricating artificial nanopores in solid state materials. These methods, however, which are capable of producing only pores in membranes are not capable of producing longer channels needed to achieve true single-molecule sequencing of long biological polymers such as DNA or RNA.

Accordingly, there is a need in the field for devices capable of yielding sequence and other information for long biological polymers such as DNA or RNA.

SUMMARY OF THE INVENTION

In meeting the described challenges, in a first aspect the present invention provides methods for characterizing one or more features of a macromolecule, comprising linearizing a macromolecule residing at least in part within a nanochannel, at least a portion of the nanochannel being capable of physically constraining at least a portion of the macromolecule so as to maintain in linear form that portion of the macromolecule, and the nanochannel comprising at least one constriction; transporting at least a portion of the macromolecule within at least a portion of the nanochannel such that at least a portion of the macromolecule passes through the constriction; monitoring at least one signal arising in connection with the macromolecule passing through the constriction; and correlating the at least one signal to one or more features of the macromolecule.

In a second aspect, the present invention provides devices for analyzing a linearized macromolecule, comprising two or more fluid reservoirs; and a nanochannel comprising a constriction, the nanochannel placing the at least two fluid reservoirs in fluid communication with one another.

Further provided are methods for transporting a macromolecule, comprising providing at least two fluid reservoirs; providing an at least partially linearized macromolecule, at least a portion of the macromolecule residing in a nanochannel, the nanochannel placing the at least two reservoirs in fluid communication with one another, the nanochannel comprising a constriction; and applying a gradient to the macromolecule, the gradient giving rise to at least a portion of the linearized macromolecule being transported within at least a portion of the nanochannel.

Additionally provided are methods for fabricating a constricted nanochannel, comprising providing a nanochannel; the nanochannel having an internal diameter in the range of from about 0.5 nm to about 1000 nm, and the nanochannel having a length of at least about 10 nm; reducing the internal diameter of the nanochannel either at a location within the nanochannel, at a location proximate to the end of the nanochannel, or both, so as to give rise to a constriction within or adjacent to the nanochannel, the constriction having an internal diameter in fluidic communication with the nanochannel, the nanochannel being capable of maintaining a linearized macromolecule in its linearized form, and the reduced internal diameter being capable of permitting the passage of at least a portion of a linearized macromolecule.

Also disclosed are methods for linearizing a macromolecule, comprising placing a macromolecule in a nanochannel, at least a portion of the nanochannel being capable of physically constraining at least a portion of the macromolecule so as to maintain in linear form that portion of the macromolecule.

The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.

