We have fabricated and characterized a novel DNA based nanofiber created by synergistic self-assembly of guanine quadruplex (Hoogsteen base pairing) and double-stranded DNA (Watson-Crick base pairing). The assembly of our nanofibers occurs in several steps. First, duplexes are generated from two DNA strands that each contains a long sequence of guanines, complementary sequences that flank the G-rich region, and single-stranded overhangs. Addition of potassium ions drives quadruplex formation of preformed duplexes, a method that has been termed synapsable quadruplex formation. Incubation of preformed duplexes in potassium-containing buffer creates quadruplexes in addition to larger molecular mass species that we hypothesize are linked quadruplexes. In addition to observation of these larger species by gel electrophoresis, solutions were analyzed by atomic force microscopy and revealed nanofibers. Analysis of the atomic force microscopy images indicates that fibers form with lengths ranging from 250 nm to 2000 nm and heights from 0.8 nm to 4.0 nm. This work is a first step toward the creation of new structurally heterogeneous (quadruplex/duplex), yet controllable, DNA based materials exhibiting novel properties suitable for a diverse array of nanotechnology applications.